Opsin-binding ligands, compositions and methods of use

ABSTRACT

Compounds are disclosed that are useful for treating ophthalmic conditions caused by or related to production of toxic visual cycle products that accumulate in the eye, such as dry adult macular degeneration, as well as conditions caused by or related to the misfolding of mutant opsin proteins and/or the mis-localization of opsin proteins. Compositions of these compounds alone or in combination with other therapeutic agents are also described, along with therapeutic methods of using such compounds and/or compositions. Methods of synthesizing such agents are also disclosed.

This application claims priority of U.S. Provisional Application Ser.No. 61/564,401, filed 29 Nov. 2011, Ser. No. 61/561,434, filed 18 Nov.2011, and Ser. No. 61/627,855, filed 19 Oct. 2011, the disclosures ofall of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compounds and compositions thereof foruse in the treatment and/or prevention of ophthalmic diseases as well asmethods of using such compounds and/or compositions.

BACKGROUND OF THE INVENTION

A diminished visual acuity or total loss of vision may result from anumber of eye diseases or disorders caused by dysfunction of tissues orstructures in the anterior segment of the eye and/or posterior segmentof the eye. Of those that occur as a consequence of a dysfunction in theanterior segment, aberrations in the visual cycle are often involved.The visual cycle (also frequently referred to as the retinoid cycle)comprises a series of light-driven and/or enzyme catalyzed reactionswhereby a light-sensitive chromophore (called rhodopsin) is formed bycovalent bonding between the protein opsin and the retinoid agent11-cis-retinal and subsequently, upon exposure to light, the11-cis-retinal is converted to all-trans-retinal, which can then beregenerated into 11-cis-retinal to again interact with opsin. A numberof visual, ophthalmic, problems can arise due to interference with thiscycle. It is now understood that at least some of these problems are dueto improper protein folding, such as that of the protein opsin.

The main light and dark photoreceptor in the mammalian eye is the rodcell, which contains a folded membrane containing protein molecules thatcan be sensitive to light, the main one being opsin. Like other proteinspresent in mammalian cells, opsin is synthesized in the endoplasmicreticulum (i.e., on ribosomes) of the cytoplasm and then conducted tothe cell membrane of rod cells. In some cases, such as due to geneticdefects and mutation of the opsin protein, opsin can exhibit improperfolding to form a conformation that either fails to properly insert intothe membrane of the rod cell or else inserts but then fails to properlyreact with 11-cis-retinal to form native rhodopsin. In either case, theresult is moderate to severe interference with visual perception in theanimal so afflicted.

Among the diseases and conditions linked to improper opsin folding isretinitis pigmentosa (RP), a progressive ocular-neurodegenerativedisease (or group of diseases) that affects an estimated 1 to 2 millionpeople worldwide. In RP, photoreceptor cells in the retina are damagedor destroyed, leading to loss of peripheral vision (i.e., tunnel vision)and subsequent partial or near-total blindness.

In the American population the most common defect occurs as a result ofreplacement of a proline residue by a histidine residue at amino acidnumber 23 in the opsin polypeptide chain (dubbed “P23H”), caused by amutation in the gene for opsin. The result is production of adestabilized form of the protein, which is misfolded and aggregates inthe cytoplasm rather than being transported to the cell surface. Likemany other protein conformational diseases (PCDs), the clinically commonP23H opsin mutant associated with autosomal dominant RP is misfolded andretained intracellularly. The aggregation of the misfolded protein isbelieved to result in photoreceptor damage and cell death.

Recent studies have identified small molecules that stabilize misfoldedmutant proteins associated with disease. Some of these, dubbed “chemicalchaperones,” stabilize proteins non-specifically. Examples of theseinclude glycerol and trimethylamine oxide. These are not very desirablefor treating ophthalmic disease because such treatment usually requireshigh dosages that may cause toxic side effects. Other agents, dubbed“pharmacological chaperones,” (which include native ligands andsubstrate analogs) act to stabilize the protein by binding to specificsites and have been identified for many misfolded proteins, e.g.,G-protein coupled receptors. Opsin is an example of a G-protein coupledreceptor and its canonical pharmacological chaperones include the classof compounds referred to as retinoids. Thus, certain retinoid compoundshave been shown to stabilize mutant opsin proteins (see, for example,U.S. Patent Pub. 2004-0242704, as well as Noorwez et al., J. Biol.Chem., 279(16): 16278-16284 (2004)).

The visual cycle comprises a series of enzyme catalyzed reactions,usually initiated by a light impulse, whereby the visual chromophore ofrhodopsin, consisting of opsin protein bound covalently to11-cis-retinal, is converted to an all-trans-isomer that is subsequentlyreleased from the activated rhodopsin to form opsin and theall-trans-retinal product. This part of the visual cycle occurs in theouter portion of the rod cells of the retina of the eye. Subsequentparts of the cycle occur in the retinal pigmented epithelium (RPE).Components of this cycle include various enzymes, such as dehydrogenasesand isomerases, as well as transport proteins for conveying materialsbetween the RPE and the rod cells.

As a result of the visual cycle, various products are produced, calledvisual cycle products. One of these is all-trans-retinal produced in therod cells as a direct result of light impulses contacting the11-cis-retinal moiety of rhodopsin. All-trans-retinal, after releasefrom the activated rhodopsin, can be regenerated back into11-cis-retinal or can react with an additional molecule ofall-trans-retinal and a molecule of phosphatidylethanolamine to produceN-retinylidene-N-retinylethanolamine (dubbed “A2E”), an orange-emittingfluorophore that can subsequently collect in the rod cells and in theretina pigmented epithelium (RPE). As A2E builds up (as a normalconsequence of the visual cycle) it can also be converted intolipofuscin, a toxic substance that has been implicated in severalabnormalities, including ophthalmic conditions such as wet and dry agerelated macular degeneration (ARMD). A2E can also prove toxic to the RPEand has been associated with dry ARMD.

Because the build-up of toxic visual cycle products is a normal part ofthe physiological process, it is likely that all mammals, especially allhumans, possess such an accumulation to some extent throughout life.However, during surgical procedures on the eye, especially on theretina, where strong light is required over an extended period, forexample, near the end of cataract surgery and while implanting the newlens, these otherwise natural processes can cause toxicity because ofthe build-up of natural products of the visual cycle. Additionally,excessive rhodopsin activation as a result of bright light stimulationcan cause photoreceptor cell apoptosis via an AP-1 transcription factordependent mechanism. Because of this, there is a need for agents thatcan be administered prior to, during or after (or any combination ofthese) the surgical process and that has the effect of inhibitingrhodopsin activation as well as reducing the production of visual cycleproducts that would otherwise accumulate and result in toxicity to theeye, especially to the retina.

The present invention answers this need by providing small moleculeswhich noncovalently bind to opsin or mutated forms of opsin for treatingand/or amelioration such conditions, if not preventing them completely.Importantly, such agents are not natural retinoids and thus are nottightly controlled for entrance into the rod cells, where mutated formsof opsin are synthesized and/or visual cycle products otherwiseaccumulate. Therefore, such agents can essentially be titrated in asneeded for facilitating the proper folding trafficking of mutated opsinsto the cell membrane or prevention of rhodopsin activation that can leadto the excessive build-up of visual cycle products likeall-trans-retinal that in turn can lead to toxic metabolic products.Such compounds may compete with 11-cis-retinal to reduceall-trans-retinal by tying up the retinal binding pocket of opsin toprevent excessive all-trans-retinal build up. Thus, the compoundsprovided by the present invention have the advantage that they do notdirectly inhibit the enzymatic processes by which 11-cis-retinal isproduced in the eye (thus not contributing to retinal degeneration).Instead, the formation of all-trans-retinal is limited and thereby theformation of A2E is reduced. Finally, by limiting the ability of11-cis-retinal to combine with opsin to form rhodopsin, rhodopsinactivation caused by bright light stimulation especially duringophthalmic surgery is also diminished thus preventing the photocelldeath that results.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Inboth cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision. The present invention solves this problem by providing amethod of correcting mislocalized opsin within a photoreceptor cell bycontacting a mislocalized opsin protein with an opsin-binding agent thatbinds reversibly and/or non-covalently to said mislocalized opsinprotein, and promotes the appropriate intracellular processing andtransport of said opsin protein. This correction of mislocalizationrelieves photoreceptor cell stress, preventing decline in viability anddeath of photoreceptor cells in various diseases of vision loss, and innormal age-related decline in dim-light and peripheral rod-mediatedvision, central cone-mediated vision, and loss of night vision.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds having thestructure of Formula I, including pharmaceutically acceptable salts,solvates and hydrates thereof, and compositions of said compounds:

wherein R¹, R², R_(a), R_(b) and A

B

C

D

E are as described elsewhere herein.

In a related aspect, the present invention relates to a method ofinhibiting the formation or accumulation of a visual cycle product,comprising contacting an opsin protein with a compound recited herein toinhibit formation of said visual cycle product relative to when saidcontacting does not occur.

In a further aspect, the present invention relates to a method to reducethe light toxicity associated with ophthalmic surgery by preventingrhodopsin regeneration during surgery to a mammalian eye and/or preventor slow the formation of toxic visual cycle products by fractionallypreventing rhodopsin formation during periods of light activationthereby providing a treatment of ocular conditions associated with thebuild up of visual products such as wet or dry ARMD.

In yet a further aspect, the present invention relates to a method ofcorrecting the proper folding and trafficking of mutated opsin proteins,comprising contacting a mutated opsin protein with a compound thatstabilizes the proper three dimensional conformation of the proteinrelative to when said contacting does not occur wherein the compound hasthe structure of Formula I including pharmaceutically acceptable salts,solvates and hydrates thereof.

In one embodiment, the ligand selectively binds reversibly ornon-covalently to opsin. In another embodiment, the ligand binds at ornear the 11-cis-retinal binding pocket of the opsin protein. In yetanother embodiment, the ligand binds to the opsin protein so as toinhibit or slow the covalent binding of 11-cis-retinal to the opsinprotein when the 11-cis-retinal is contacted with the opsin protein inthe presence of the ligand. In yet another embodiment, the ligand bindsto the opsin in the retinal binding pocket of opsin protein or disrupts11-cis-retinal binding to the retinal binding pocket of opsin. In yetanother embodiment, the ligand binds to the opsin protein so as toinhibit covalent binding of 11-cis-retinal to the opsin protein. In yetanother embodiment, the mammal is a human being.

In yet another embodiment, slowing or halting the progression of wet ordry ARMD is associated with reducing the level of a visual cycleproduct, for example, a visual cycle product formed fromall-trans-retinal, such as lipofuscin orN-retinylidine-N-retinylethanolamine (A2E). In yet another embodimentslowing or halting the progression of RP is associated with correctingthe folding of mutated opsins. In another embodiment, the administeringis topical administration, local administration (e.g., intraocular orperiocular injection or implant) or systemic administration (e.g., oral,injection). In yet another embodiment, the light toxicity is related toan ophthalmic procedure (e.g., ophthalmic surgery). In still anotherembodiment, the administering occurs prior to, during, or after theophthalmic surgery.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Insuch cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision. In one aspect, the invention provides a method ofcorrecting mislocalized opsin within a photoreceptor cell, comprisingcontacting a mislocalized opsin protein with an opsin-binding agent thatbinds reversibly and/or non-covalently to said mislocalized opsinprotein to promote the appropriate intracellular processing andtransport of said opsin protein. This correction of mislocalizationreduces photoreceptor cell stress, preventing photoreceptor cell declinein viability and death in various diseases of vision loss, and in normalage-related decline in dim-light and peripheral rod-mediated vision,central cone-mediated vision, and loss of night vision.

In various embodiments, the ocular protein mislocalization disorder isany one or more of wet or dry form of macular degeneration, retinitispigmentosa, a retinal or macular dystrophy, Stargardt's disease,Sorsby's dystrophy, autosomal dominant drusen, Best's dystrophy,peripherin mutation associate with macular dystrophy, dominant form ofStargardt's disease, North Carolina macular dystrophy, light toxicity,retinitis pigmentosa, normal vision loss related aging and normal lossof night vision related to aging.

In still another embodiment, the method further involves administeringto a mammal, preferably a human being, an effective amount of at leastone additional agent selected from the group consisting of a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp90 chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, anda histone deacetylase inhibitor. In yet another embodiment, the opsinbinding ligand and the additional agent are administered simultaneously.

In still another embodiment, the opsin binding ligand and the additionalagent are each incorporated into a composition that provides for theirlong-term release. In another embodiment, the composition is part of amicrosphere, nanosphere, nano emulsion or implant. In anotherembodiment, the composition further involves administering a mineralsupplement, at least one anti-inflammatory agent, such as a steroid(e.g., any one or more of cortisone, hydrocortisone, prednisone,prednisolone, methylprednisolone, triamcinolone, betamethasone,beclamethasone and dexamethasone), or at least one anti-oxidant, such asvitamin A, vitamin C and vitamin E. In various embodiments, the opsinbinding ligand, the anti-inflammatory agent, and/or the anti-oxidant areadministered simultaneously.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 □Mof □-ionone during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

DEFINITIONS

As used throughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings.

By “mislocalization” of a photoreceptor cell visual pigment protein (forexample, opsin, especially human opsin) is meant that the synthesizedprotein is not found at the normal or appropriate cellular location.

“Pharmacologic chaperones” refer to small molecular weight chemicalcompounds that interact with a protein (usually with a misfolded, orunfolded protein) in such a way as to alter the folding or confirmationof said protein. Such an interaction can have diverse consequences onthe cellular fate of the protein, including but not limited to leadingto increased stability and increased levels of functional protein,increased stability and increased levels of non-functional protein, ordecreased stability and decreased levels of functional or non-functionalprotein.

“Productive chaperone” refers to a pharmacologic chaperone that wheninteracting with a protein leads to an increased level of functionalprotein.

“Counterproductive; shipwreck or destructive chaperone” refers to apharmacologic chaperone that interacts with a protein (usually with amis-folded, or un-folded protein) and this interaction leads to adecreased stability and/or decreased levels of functional ornon-functional protein.

By “proteasomal inhibitor” is meant a compound that reduces aproteasomal activity, such as the degradation of a ubiquinated protein.

By “autophagy inhibitor” is meant a compound that reduces thedegradation of a cellular component by a cell in which the component islocated.

By “lysosomal inhibitor” is meant a compound that reduces theintracellular digestion of macromolecules by a lysosome. In oneembodiment, a lysosomal inhibitor decreases the proteolytic activity ofa lysosome.

By “Inhibitor of ER-Golgi protein transport” is meant a compound thatreduces the transport of a protein from the ER (endoplasmic reticulum)to the Golgi, or from the Golgi to the ER.

By “HSP90 chaperone inhibitor” is meant a compound that reduces thechaperone activity of heat shock protein 90 (HSP90). In one embodiment,the inhibitor alters protein binding to an HSP90 ATP/ADP pocket.

By “heat shock response activator” is meant a compound that increasesthe chaperone activity or expression of a heat shock pathway component.Heat shock pathway components include, but are not limited to, HSP100,HSP90, HSP70, HASP60, HSP40 and small HSP family members.

By “glycosidase inhibitor” is meant a compound that reduces the activityof an enzyme that cleaves a glycosidic bond.

By “histone deacetylase inhibitor” is meant a compound that reduces theactivity of an enzyme that deacetylates a histone.

By “reduces” or “increases” is meant a negative or positive alteration,respectively. In particular embodiments, the alteration is by at leastabout 10%, 25%, 50%, 75%, or 100% of the initial level of the proteinproduced in the absence of the opsin binding ligand.

As used herein, the term “wild-type conformation” refers to the threedimensional conformation or shape of a protein that is free of mutationsto its amino acid sequence. For opsin, this means a protein free frommutations that cause misfiling, such as the mutation designated P23H(meaning that a proline is replaced by a histidine at residue 23starting from the N-terminus). Opsin in a “wild-type conformation” iscapable of opsin biological function, including but not limited to,retinoid binding, visual cycle function, and insertion into aphotoreceptor membrane.

By “agent” is meant a small compound (also called a “compound”),polypeptide, polynucleotide, or fragment thereof. The terms compound andagent are used interchangeably unless specifically stated otherwiseherein for a particular agent or compound.

By “correcting the conformation” of a protein is meant inducing theprotein to assume a conformation having at least one biological activityassociated with a wild-type protein.

By “misfolded opsin protein” is meant a protein whose tertiary structurediffers from the conformation of a wild-type protein, such that themisfolded protein lacks one or more biological activities associatedwith the wild-type protein.

By “selectively binds” is meant a compound that recognizes and binds apolypeptide of the invention, such as opsin, but which does notsubstantially recognize and bind other molecules, especially non-opsinpolypeptides, in a sample, for example, a biological sample.

By “effective amount” or “therapeutically effective amount” is meant alevel of an agent sufficient to exert a physiological effect on a cell,tissue, or organ or a patient. As used herein, it is the amountsufficient to effect the methods of the invention to achieve the desiredresult.

By “pharmacological chaperone” is meant a molecule that upon contactinga mutant protein is able to facilitate/stabilize the proper folding ofthe protein such that it acts and functions much more like wild typeprotein than would be the case in the absence of the molecule.

By “control” is meant a reference condition. For example, where a cellcontacted with an agent of the invention is compared to a correspondingcell not contacted with the agent, the latter is the “control” or“control” cell.

By “treat” is meant decrease, suppress, attenuate, diminish, arrest, orstabilize the development or progression of a disease, preferably anocular disease, such as RP, AMD and/or light toxicity.

By “prevent” is meant reduce the risk that a subject will develop acondition, disease, or disorder, preferably an ocular disease, such asRP, AMD and/or light toxicity.

By “competes for binding” is meant that a compound of the invention andan endogenous ligand are incapable of binding to a target at the sametime. Assays to measure competitive binding are known in the art, andinclude, measuring a dose dependent inhibition in binding of a compoundof the invention and an endogenous ligand by measuring t_(1/2), forexample.

A “pharmaceutically acceptable salt” is a salt formed from an acid or abasic group of one of the compounds of the invention. Illustrative saltsinclude, but are not limited to, sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbatc, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesuifonate, and pamoate (i.e.,1,1′-methytene-bis-(2-hydroxy-3-naphthoate)) salts.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound of the invention having an acidic functionalgroup, such as a carboxylic acid functional group, and apharmaceutically acceptable inorganic or organic base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine;tributyl amine; pyridine; N-methyl-N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), suchas mono-, bis-, or tris-(2-hydroxyethyl)-amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound disclosed herein, e.g., a salt of a compound ofExample 1, having a basic functional group, such as an amino functionalgroup, and a pharmaceutically acceptable inorganic or organic acid.Suitable acids include, but are not limited to, hydrogen sulfate, citricacid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide,hydrogen iodide, nitric acid,

phosphoric acid, isonicotinic acid, lactic acid, salicylic acid,tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid,fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formicacid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, and p-toluenesulfonic acid.

The term “pharmaceutically-acceptable excipient” as used herein meansone or more compatible solid or liquid tiller, diluents or encapsulatingsubstances that are suitable for administration into a human. The term“excipient” includes an inert substance added to a pharmacologicalcomposition to further facilitate administration of a compound. Examplesof excipients include but are not limited to calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, vegetable oils and polyethylene glycols.

The term “carrier” denotes an organic or inorganic ingredient, naturalor to synthetic, with which the active ingredient is combined tofacilitate administration.

The term “parenteral” includes subcutaneous, intrathecal, intravenous,intramuscular, intraperitoneal, or infusion.

The term “visual cycle product” refers to a chemical entity produced asa natural product of one or more reactions of the visual cycle (thereactive cycle whereby opsin protein binds 11-cis-retinal to formrhodopsin, which accepts a light impulse to convert 11-cis-retinal toall trans-retinal, which is then released from the molecule, toregenerate opsin protein with subsequent binding of a new 11-cis-retinalto regenerate rhodopsin). Such visual cycle products include, but arenot limited to, all-trans-retinal, lipofuscin and A2E.

The term “light toxicity” refers to any condition affecting vision thatis associated with, related to, or caused by the production and/oraccumulation of visual cycle products. Visual cycle products include,but are not limited to, all-trans-retinal, lipofuscin or A2E. In oneparticular embodiment, light toxicity is related to exposure of the eyeto large amounts of light or to very high light intensity, occurring,for example, during a surgical procedure on the retina.

The term “opsin” refers to an opsin protein, preferably a mammalianopsin protein, most preferably a human opsin protein. In one embodiment,the opsin protein is in the wild-type (i.e., physiologically active)conformation. One method of assaying for physiological activity isassaying the ability of opsin to bind 11-cis-retinal and form activerhodopsin. A mutant opsin, such as the P23H mutant, that is ordinarilymisfolded has a reduced ability to bind 11-cis-retinal, and thereforeforms little or no rhodopsin. Where the conformation of the mutant opsinhas been corrected (for example, by binding to a pharmacologicalchaperone), the opsin is correctly inserted into the rod cell membraneso that its conformation is the same, or substantially the same, as thatof a non-mutant opsin. This allows the mutant opsin to bind11-cis-retinal to form active rhodopsin. Therefore, the methods of theinvention operate to reduce the formation of visual cycle products.

“Alkyl” refers to an unbroken non-cyclic chain of carbon atoms that maybe substituted with other chemical groups. It may also be branched orunbranched, substituted or unsubstituted.

“Lower alkyl” refers to a branched or straight chain acyclic alkyl groupcomprising one to ten carbon atoms, preferably one to eight carbonatoms, more preferably one to six carbon atoms. Exemplary lower alkylgroups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, pentyl, neopentyl, iso-amyl, hexyl, and octyl.

All alkyl, alkenyl or alkynyl groups disclosed herein may be substitutedwith one or more of the following: lower alkyl, hydroxy, ester, amidyl,oxo, carboxyl, carboxamido, halo, cyano, nitrate, nitrite, thionitrate,thionitrite sulfhydryl and amino groups (as elsewhere defined herein).

“Haloalkyl” refers to a lower alkyl group, an alkenyl group, an alkynylgroup, a bridged cycloalkyl group, a cycloalkyl group or a heterocyclicring, as defined herein, to which is appended one or more halogens, asdefined herein. Exemplary haloalkyl groups include trifluoromethyl,chloromethyl, 2-bromobutyl and 1-bromo-2-chloro-pentyl.

“Alkenyl” refers to a branched or straight chain C₂-C₁₀ hydrocarbon(preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₈ hydrocarbon)that can comprise one or more carbon-carbon double bonds. Exemplaryalkenyl groups include propylenyl, buten-1-yl, isobutenyl, penten-1-yl,2,2-methylbuten-1-yl, 3-methylbuten-1-yl, hexan-1-yl, hepten-1-yl andocten-1-yl.

“Lower alkenyl” refers to a branched or straight chain C₂-C₄ hydrocarbonthat can comprise one or two carbon-carbon double bonds.

“Substituted alkenyl” refers to a branched or straight chain C₂-C₁₀hydrocarbon (preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₆hydrocarbon) which can comprise one or more carbon-carbon double bonds,wherein one or more of the hydrogen atoms have been replaced with one ormore R¹⁰⁰ groups, wherein each R¹⁰⁰ is independently a hydroxy, an oxo,a carboxyl, a carboxamido, a halo, a cyano or an amino group, as definedherein.

“Alkynyl” refers to an unsaturated acyclic C₂-C₁₀ hydrocarbon(preferably a C₂-C₈ hydrocarbon′, more preferably a C₂-C₆ hydrocarbon)that can comprise one or more carbon-carbon triple bonds. Exemplaryalkynyl groups include ethynyl, propynyl, butyn-1-yl, butyn-2-yl,pentyl-1-yl, pentyl-2-yl, 3-methylbutyn-1-yl, hexyl-1-yl, hexyl-2-yl,hexyl-3-yl and 3,3-dimethyl-butyn-1-yl.

“Lower alkynyl” refers to a branched or straight chain C₂-C₄ hydrocarbonthat can comprise one or two carbon-carbon triple bonds

“Bridged cycloalkyl” refers to two or more cycloalkyl groups,heterocyclic groups, or a combination thereof fused via adjacent ornon-adjacent atoms. Bridged cycloalkyl groups can be unsubstituted orsubstituted with one, two or three substituents independently selectedfrom alkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, halo,carboxyl, alkylcarboxylic acid, aryl, amidyl, ester, alkylcarboxylicester, carboxamido, alkylcarboxamido, oxo and nitro. Exemplary bridgedcycloalkyl groups include adamantyl, decahydronapthyl, quinuclidyl,2,6-dioxabicyclo(3.3.0)octane, 7-oxabicyclo(2.2.1)heptyl and8-azabicyclo(3,2,1)oct-2-enyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarboncomprising from about 3 to about 10 carbon atoms. Cycloalkyl groups canbe unsubstituted or substituted with one, two or three substituentsindependently selected from alkyl, alkoxy, amino, alkylamino,dialkylamino, arylamino, diarylamino, alkylarylamino, aryl, amidyl,ester, hydroxy, halo, carboxyl, alkylcarboxylic acid, alkylcarboxylicester, carboxamido, alkylcarboxamido, oxo, alkylsulfinyl, and nitro.Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl and cyclohepta-1,3-dienyl.

“Heterocyclic ring or group” refers to a saturated or unsaturated cyclicor polycyclic hydrocarbon group having about 2 to about 12 carbon atomswhere 1 to about 4 carbon atoms are replaced by one or more nitrogen,oxygen and/or sulfur atoms. Sulfur may be in the thio, sulfinyl orsulfonyl oxidation state. The heterocyclic ring or group can be fused toan aromatic hydrocarbon group. Heterocyclic groups can be unsubstitutedor substituted with one, two or three substituents independentlyselected from alkyl, alkoxy, amino, alkylthio, aryloxy, arylthio,arylalkyl, hydroxy, oxo, thial, halo, carboxyl, carboxylic ester,alkylcarboxylic acid, alkylcarboxylic ester, aryl, arylcarboxylic acid,arylcarboxylic ester, amidyl, ester, alkylcarbonyl, arylcarbonyl,alkylsulfinyl, carboxamido, alkylcarboxamido, arylcarboxamido, sulfonicacid, sulfonic ester, sulfonamide nitrate and nitro. Exemplaryheterocyclic groups include pyrrolyl, furyl, thienyl,3-pyrrolinyl,4,5,6-trihydro-2H-pyranyl, pyridinyl, 1,4-dihydropyridinyl,pyrazolyl, triazolyl, pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl,thieno[2,3-d]pyrimidine, 4,5,6,7-tetrahydrobenzo[b]thiophene,imidazolyl, indolyl, thiophenyl, furanyl, tetrahydrofuranyl, tetrazolyl,pyrrolinyl, pyrrolindinyl, oxazolindinyl 1,3-dioxolanyl, imidazolinyl,imidazolindinyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl,1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl,4H-pyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl,thiomorpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl,1,3,5-trithianyl, benzo(b)thiophenyl, benzimidazolyl, benzothiazolinyl,quinolinyl and 2,6-dioxabicyclo(3.3.0)octane.

“Heterocyclic compounds” refer to mono- and polycyclic compoundscomprising at least one aryl or heterocyclic ring.

“Aryl” refers to a monocyclic, bicyclic, carbocyclic or heterocyclicring system comprising one or two aromatic rings. Exemplary aryl groupsinclude phenyl, pyridyl, napthyl, quinoyl, tetrahydronaphthyl, furanyl,indanyl, indenyl, indoyl. Aryl groups (including bicyclic aryl groups)can be unsubstituted or substituted with one, two or three substituentsindependently selected from alkyl, alkoxy, alkylthio, amino, alkylamino,dialkylamino, arylamino, diarylamino, alkylarylamino, halo, cyano,alkylsulfinyl, hydroxy, carboxyl, carboxylic ester, alkylcarboxylicacid, alkylcarboxylic ester, aryl, arylcarboxylic acid, arylcarboxylicester, alkylcarbonyl, arylcarbonyl, amidyl, ester, carboxamido,alkylcarboxamido, carbomyl, sulfonic acid, sulfonic ester, sulfonamidoand nitro. Exemplary substituted aryl groups include tetrafluorophenyl,pentafluorophenyl, sulfonamide, alkylsulfonyl and arylsulfonyl.

“Cycloalkenyl” refers to an unsaturated cyclic C₃-C₁₀ hydrocarbon(preferably a C₃-C₈ hydrocarbon, more preferably a C₃-C₆ hydrocarbon),which can comprise one or more carbon-carbon double bonds.

“Alkylaryl” refers to an alkyl group, as defined herein, to which isappended an aryl group, as defined herein. Exemplary alkylaryl groupsinclude benzyl, phenylethyl, hydroxybenzyl, fluorobenzyl andfluorophenylethyl.

“Arylalkyl” refers to an aryl radical, as defined herein, attached to analkyl radical, as defined herein. Exemplary arylalkyl groups includebenzyl, phenylethyl, 4-hydroxybenzyl, 3-fluorobenzyl and2-fluorophenylethyl.

“Arylalkenyl” refers to an aryl radical, as defined herein, attached toan alkenyl radical, as defined herein. Exemplary arylalkenyl groupsinclude styryl and propenylphenyl.

“Cycloalkylalkyl” refers to a cycloalkyl radical, as defined herein,attached to an alkyl radical, as defined herein.

“Cycloalkylalkoxy” refers to a cycloalkyl radical, as defined herein,attached to an alkoxy radical, as defined herein.

“Cycloalkylalkylthio” refers to a cycloalkyl radical, as defined herein,attached to an alkylthio radical, as defined herein.

“Heterocyclicalkyl” refers to a heterocyclic ring radical, as definedherein, attached to an alkyl radical, as defined herein.

“Arylheterocyclic ring” refers to a bi- or tricyclic ring comprised ofan aryl ring, as defined herein, appended via two adjacent carbon atomsof the aryl ring to a heterocyclic ring, as defined herein. Exemplaryarylheterocyclic rings include dihydroindole and1,2,3,4-tetra-hydroquinoline.

“Alkylheterocyclic ring” refers to a heterocyclic ring radical, asdefined herein, attached to an alkyl radical, as defined herein.Exemplary alkylheterocyclic rings include 2-pyridylmethyl and1-methylpiperidin-2-one-3-methyl.

“Alkoxy” refers to R₅₀O—, wherein R₅₀ is an alkyl group, an alkenylgroup or an alkynyl group as defined herein (preferably a lower alkylgroup or a haloalkyl group, as defined herein). Exemplary alkoxy groupsinclude methoxy, ethoxy, t-butoxy, cyclopentyloxy, trifluoromethoxy,propenyloxy and propargyloxy.

“Aryloxy” refers to R₅₅O—, wherein R₅₅ is an aryl group, as definedherein. Exemplary arylkoxy groups include phenoxy, napthyloxy,quinolyloxy, isoquinolizinyloxy.

“Alkylthio” refers to R₅₀S—, wherein R₅₀ is an alkyl group, as definedherein.

“Lower alkylthio” refers to a lower alkyl group, as defined herein,appended to a thio group, as defined herein.

“Arylalkoxy” or “alkoxyaryl” refers to an alkoxy group, as definedherein, to which is appended an aryl group, as defined herein. Exemplaryarylalkoxy groups include benzyloxy, phenylethoxy andchlorophenylethoxy.

“Arylalklythio” refers to an alkylthio group, as defined herein, towhich is appended an aryl group, as defined herein. Exemplaryarylalklythio groups include benzylthio, phenylethylthio andchlorophenylethylthio.

“Arylalkylthioalkyl” refers to an arylalkylthio group, as definedherein, to which is appended an alkyl group, as defined herein.Exemplary arylalklythioalkyl groups include benzylthiomethyl,phenylethylthiomethyl and chlorophenylethylthioethyl.

“Alkylthioalkyl” refers to an alkylthio group, as defined herein, towhich is appended an alkyl group, as defined herein. Exemplaryalkylthioalkyl groups include allylthiomethyl, ethylthiomethyl andtrifluoroethylthiomethyl.

“Alkoxyalkyl” refers to an alkoxy group, as defined herein, appended toan alkyl group, as defined herein. Exemplary alkoxyalkyl groups includemethoxymethyl, methoxyethyl and isopropoxymethyl.

“Alkoxyhaloalkyl” refers to an alkoxy group, as defined herein, appendedto a haloalkyl group, as defined herein. Exemplary alkoxyhaloalkylgroups include 4-methoxy-2-chlorobutyl.

“Cycloalkoxy” refers to R₅₄O—, wherein R₅₄ is a cycloalkyl group or abridged cycloalkyl group, as defined herein. Exemplary cycloalkoxygroups include cyclopropyloxy, cyclopentyloxy and cyclohexyloxy.

“Cycloalkylthio” refers to R₅₄S—, wherein R₅₄ is a cycloalkyl group or abridged cycloalkyl group, as defined herein. Exemplary cycloalkylthiogroups include cyclopropylthio, cyclopentylthio and cyclohexylthio.

“Haloalkoxy” refers to an alkoxy group, as defined herein, in which oneor more of the hydrogen atoms on the alkoxy group are substituted withhalogens, as defined herein. Exemplary haloalkoxy groups include1,1,1-trichloroethoxy and 2-bromobutoxy.

“Hydroxy” refers to —OH. “Oxy” refers to —O—. “Oxo” refers to ═O.

“Oxylate” refers to —O⁻ R₇₇ ⁺ wherein R₇₇ is an organic or inorganiccation.

“Thiol” refers to —SH. “Thio” refers to —S—.

“Oxime” refers to ═N—OR₈₁ wherein R₈₁ is a hydrogen, an alkyl group, anaryl group, an alkylsulfonyl group, an arylsulfonyl group, a carboxylicester, an alkylcarbonyl group, an arylcarbonyl group, a carboxamidogroup, an alkoxyalkyl group or an alkoxyaryl group.

“Hydrazone” refers to ═N—N(R₈₁)(R₈₁) wherein R′₈₁ is independentlyselected from R₈₁, and R₈₁ is as defined herein.

“Hydrazino” refers to H₂N—N(H)—.

“Organic cation” refers to a positively charged organic ion. Exemplaryorganic cations include alkyl substituted ammonium cations.

“Inorganic cation” refers to a positively charged metal ion. Exemplaryinorganic cations include Group I metal cations such as for example,sodium, potassium, magnesium and calcium.

“Hydroxyalkyl” refers to a hydroxy group, as defined herein, appended toan alkyl group, as defined herein.

“Nitrate” refers to —O—NO₂ i.e. oxidized nitrogen.

“Nitro” refers to the group —NO₂ and “nitrosated” refers to compoundsthat have been substituted therewith. “Nitrile” and “cyano” refer to—CN.

“Halogen” or “halo” refers to iodine (I), bromine (Br), chlorine (Cl),and/or fluorine (F).

“Imine” refers to —C(═N—R₅₁)— wherein R₅₁ is a hydrogen atom, an alkylgroup, an aryl group or an arylheterocyclic ring, as defined herein.

“Amine” refers to any organic compound that contains at least one basicnitrogen atom.

“Amino” refers to —NH₂, an alkylamino group, a dialkylamino group, anarylamino group, a diarylamino group, an alkylarylamino group or aheterocyclic ring, as defined herein.

“Alkylamino” refers to R₅₀NH—, wherein R₅₀ is an alkyl group, as definedherein. Exemplary alkylamino groups include methylamino, ethylamino,butylamino, and cyclohexylamino.

“Arylamino” refers to R₅₅NH—, wherein R₅₅ is an aryl group, as definedelsewhere herein.

“Dialkylamino” refers to R₅₂R₅₃N—, wherein R₅₂ and R₅₃ are eachindependently an alkyl group, as defined herein. Exemplary dialkylaminogroups include dimethylamino, diethylamino and methyl propargylamino.

“Diarylamino” refers to R₅₅R₆₀N—, wherein R₅₅ and R₆₀ are eachindependently an aryl group, as defined herein.

“Alkylarylamino” or “arylalkylamino” refers to R₅₂R₅₅N—, wherein R₅₂ isan alkyl group, as defined herein, and R₅₅ is an aryl group, as definedherein.

“Alkylarylalkylamino” refers to R₅₂R₇₉N—, wherein R₅₂ is an alkyl group,as defined herein, and R₇₉ is an arylalkyl group, as defined herein.

“Alkylcycloalkylamino” refers to R₅₂R₈₀N—, wherein R₅₂ is an alkylgroup, as defined herein, and R₈₀ is a cycloalkyl group, as definedherein.

“Aminoalkyl” refers to an amino group, an alkylamino group, adialkylamino group, an arylamino group, a diarylamino group, analkylarylamino group or a heterocyclic ring, as defined herein, to whichis appended an alkyl group, as defined herein. Exemplary aminoalkylgroups include dimethylaminopropyl, diphenylaminocyclopentyl andmethylaminomethyl.

“Aminoaryl” refers to an aryl group to which is appended an alkylaminogroup, an arylamino group or an arylalkylamino group. Exemplaryaminoaryl groups include anilino, N-methylanilino and N-benzylanilino.

“Thio” refers to S—. “Sulfinyl” refers to —S(O)—.

“Methanthial” refers to —C(S)—. “Thial” refers to ═S. “Sulfonyl” refersto —S(O)₂ ⁻.

“Sulfonic acid” refers to —S(O)₂OR₇₆, wherein R₇₆ is a hydrogen, anorganic cation or an inorganic cation, as defined herein.

“Alkylsulfonic acid” refers to a sulfonic acid group, as defined herein,appended to an alkyl group, as defined herein.

“Arylsulfonic acid” refers to a sulfonic acid group, as defined herein,appended to an aryl group, as defined herein.

“Sulfonic ester” refers to —S(O)₂OR₅₈, wherein R₅₅ is an alkyl group, anaryl group, or an aryl heterocyclic ring, as defined herein.

“Sulfonamido” refers to —S(O)₂—N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ when takentogether are a heterocyclic ring, a cycloalkyl group or a bridgedcycloalkyl group, as defined herein.

“Alkylsulfonamido” refers to a sulfonamido group, as defined herein,appended to an alkyl group, as defined herein.

“Arylsulfonamido” refers to a sulfonamido group, as defined herein,appended to an aryl group, as defined herein.

“Alkylthio” refers to R₅₀S—, wherein R₅₀ is an alkyl group, as definedherein (preferably a lower alkyl group, as defined herein).

“Arylthio” refers to R₅₅S—, wherein R₅₅ is an aryl group, as definedherein.

“Arylalkylthio” refers to an aryl group, as defined herein, appended toan alkylthio group, as defined herein.

“Alkylsulfinyl” refers to R₅₀—S(O)—, wherein R₅₀ is an alkyl group, asdefined herein.

“Alkylsulfonyl” refers to R₅₀—S(O)₂—, wherein R₅₀ is an alkyl group, asdefined herein.

“Alkylsulfonyloxy” refers to R₅₀—S(O)₂—O—, wherein R₅₀ is an alkylgroup, as defined herein.

“Arylsulfinyl” refers to R₅₅—S(O)—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylsulfonyl” refers to R₅₅—S(O)₂—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylsulfonyloxy” refers to R₅₅—S(O)₂—O—, wherein R₅₅ is an aryl group,as defined herein.

“Amidyl” refers to R₅₁C(O)N(R₅₇)— wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein.

“Ester” refers to R₅₁C(O)R₈₂— wherein R₅₁ is a hydrogen atom, an alkylgroup, an aryl group or an arylheterocyclic ring, as defined herein andR₈₂ is oxygen or sulfur.

“Carbamoyl” refers to —O—C(O)N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ taken togetherare a heterocyclic ring, a cycloalkyl group or a bridged cycloalkylgroup, as defined herein.

“Carboxyl” refers to —C(O)OR₇₆, wherein R₇₆ is a hydrogen, an organiccation or an inorganic cation, as defined herein.

“Carbonyl” refers to —C(O)—.

“Alkylcarbonyl” refers to R₅₂—C(O)—, wherein R₅₂ is an alkyl group, asdefined herein.

“Arylcarbonyl” refers to R₅₅—C(O)—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylalkylcarbonyl” refers to R₅₅—R₅₂—C(O)—, wherein R₅₅ is an arylgroup, as defined herein, and R₅₂ is an alkyl group, as defined herein.

“Alkylarylcarbonyl” refers to R₅₂—R₅₅—C(O)—, wherein R₅₅ is an arylgroup, as defined herein, and R₅₂ is an alkyl group, as defined herein.

“Heterocyclicalkylcarbonyl” refer to R₇₈C(O)— wherein R₇₈ is aheterocyclicalkyl group, as defined herein.

“Carboxylic ester” refers to —C(O)OR₅₈, wherein R₅₈ is an alkyl group,an aryl group or an aryl heterocyclic ring, as defined herein.

“Alkylcarboxylic acid” and “alkylcarboxyl” refer to an alkyl group, asdefined herein, appended to a carboxyl group, as defined herein.

“Alkylcarboxylic ester” refers to an alkyl group, as defined herein,appended to a carboxylic ester group, as defined herein.

“Alkyl ester” refers to an alkyl group, as defined herein, appended toan ester group, as defined herein.

“Arylcarboxylic acid” refers to an aryl group, as defined herein,appended to a carboxyl group, as defined herein.

“Arylcarboxylic ester” and “arylcarboxyl” refer to an aryl group, asdefined herein, appended to a carboxylic ester group, as defined herein.

“Aryl ester” refers to an aryl group, as defined herein, appended to anester group, as defined herein.

“Carboxamido” refers to —C(O)N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ when takentogether are a heterocyclic ring, a cycloalkyl group or a bridgedcycloalkyl group, as defined herein.

“Alkylcarboxamido” refers to an alkyl group, as defined herein, appendedto a carboxamido group, as defined herein.

“Arylcarboxamido” refers to an aryl group, as defined herein, appendedto a carboxamido group, as defined herein.

“Urea” refers to —N(R₅₉)—C(O)N(R₅₁)(R₅₇) wherein R₅₁, R₅₇, and R₅₉ areeach independently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ taken togetherare a heterocyclic ring, a cycloalkyl group or a bridged cycloalkylgroup, as defined herein.

“Phosphoryl” refers to —P(R₇₀)(R₇₁)(R₇₂), wherein R₇₉ is a lone pair ofelectrons, thial or oxo, and R₇₁ and R₇₂ are each independently acovalent bond, a hydrogen, a lower alkyl, an alkoxy, an alkylamino, ahydroxy, an oxy or an aryl, as defined herein.

“Phosphoric′ acid” refers to —P(O)(OR₅₁)OH wherein R₅₁ is a hydrogenatom, an alkyl group, an aryl group or an arylheterocyclic ring, asdefined herein.

“Phosphinic acid” refers to —P(O)(R₅₁)OH wherein R₅₁ is a hydrogen atom,an alkyl group, an aryl group or an arylheterocyclic ring, as definedherein.

“Silyl” refers to —Si(R₇₃)(R₇₄)(R₇₅), wherein R₇₃, R₇₄ and R₇₅ are eachindependently a covalent bond, a lower alkyl, an alkoxy, an aryl or anarylalkoxy, as defined herein.

“Organic acid” refers to compound having at least one carbon atom andone or more functional groups capable of releasing a proton to a basicgroup. The organic acid preferably contains a carboxyl, a sulfonic acidor a phosphoric acid moiety. Exemplary organic acids include aceticacid, benzoic acid, citric acid, camphorsulfonic acid, methanesulfonicacid, taurocholic acid, chlordronic acid, glyphosphate and medronicacid.

“Inorganic acid” refers to a compound that does not contain at least onecarbon atom and is capable of releasing a proton to a basic group.Exemplary inorganic acids include hydrochloric acid, sulfuric acid,nitric acid and phosphoric acid.

“Organic base” refers to a carbon containing compound having one or morefunctional groups capable of accepting a proton from an acid group. Theorganic base preferably contains an amine group. Exemplary organic basesinclude triethylamine, benzyldiethylamine, dimethylethyl amine,imidazole, pyridine and pipyridine.

“Independently selected” groups are groups present in the same structurethat need not all represent the same substitution. For example, wheretwo substituents are represented as NOR_(A) and each R_(A) is said to beindependently selected from H, methyl, ethyl, etc., this means thatwhere one R_(A) is methyl, the other R_(A) may be methyl but could be Hor ethyl (or any other recited substitution).

Some of the compounds for use in the methods of the present inventionmay contain one or more chiral centers and therefore may exist inenantiomeric and diastereomeric forms. The scope of the presentinvention is intended to cover use of all isomers per se, as well asmixtures of cis and trans isomers, mixtures of diastereomers and racemicmixtures of enantiomers (optical isomers) as well. Further, it ispossible using well known techniques to separate the various forms, andsome embodiments of the invention may feature purified or enrichedspecies of a given enantiomer or diastereomer.

A “pharmacological composition” refers to a mixture of one or more ofthe compounds described herein, or pharmaceutically acceptable saltsthereof, with other chemical components, such as pharmaceuticallyacceptable carriers and/or excipients. The purpose of a pharmacologicalcomposition is to facilitate administration of a compound to anorganism.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agent fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. A physiologically acceptablecarrier should not cause significant irritation to an organism and doesnot abrogate the biological activity and properties of the administeredcompound.

A “solvate” is a complex formed by the combination of a solute (e.g., ametalloprotease inhibitor) and a solvent (e.g., water). See J. Honig etal., The Van Nostrand Chemist's Dictionary, p. 650 (1953).

The terms “optical isomer”, “geometric isomer” (e.g., a cis and/or transisomer), “stereoisomer”, and “diastereomer” have the accepted meanings(see, e.g., Hawley's Condensed Chemical Dictionary, 11th Ed.). Theillustration of specific protected forms and other derivatives of thecompounds of the instant invention is not intended to be limiting. Theapplication of other useful protecting groups, salt forms, prodrugsetc., is within the ability of the skilled artisan.

A “prodrug” is a form of a drug that must undergo chemical conversion bymetabolic processes before becoming an active, or fully active,pharmacological agent. A prodrug is not active, or is less active, inits ingested or absorbed or otherwise administered form. For example, aprodrug may be broken down by bacteria in the digestive system intoproducts, at least one of which will become active as a drug.Alternatively, it may be administered systemically, such as byintravenous injection, and subsequently be metabolized into one or moreactive molecules.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, small molecule ligands arecapable of reversibly binding non-covalently to the opsin protein andinhibiting the binding of 11-cis-retinal, to an opsin retinal bindingpocket. Such interference with retinal binding reduces the formation ofvisual cycle products, such as all-trans-retinal, and thereby inhibitsthe production of compounds such as lipofuscin and A2E with resultingreduced risk and occurrence of toxicity that can result fromaccumulation of these substances. Such compounds, acting aspharmacologic chaperones, are also able to facilitate the proper foldingand trafficking of mutant opsins associated with RP. Additionally, byinhibiting 11-cis-retinal binding and rhodopsin formation, the excessivestimulation and resulting activation of rhodopsin caused by exposure ofthe retina to bright light especially during retinal surgery reducesphotocell death.

Certain synthetic retinoids (compounds structurally related to retinol(Vitamin A alcohol)) have been reported to bind to opsin. In theembodiments of the present invention, non-retinoid small molecules(compounds having a molecular weight less than about 1000 daltons, lessthan 800, less than 600, less than 500, less than 400, or less thanabout 300 daltons) have been found to bind to opsin.

The invention features compositions and methods that are useful forreducing formation of visual cycle products and toxicity associated withthe accumulation of such products in vivo, reducing the probability ofapoptotic events associated with excessive rhodopsin activation as wellas preventing rod cell death due to aberrant processing and traffickingof mutant opsin proteins associated with RP.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Insuch cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision.

In one aspect, the invention provides a method of correctingmislocalized opsin within a photoreceptor cell, comprising contacting amislocalized opsin protein with an opsin-binding agent that bindsreversibly and/or non-covalently to said mislocalized opsin protein,thereby promoting correct intracellular processing and transport of saidopsin protein. Such opsin-binding agent is referred to as a “ProductiveChaperone.”

Such correction of mislocalization reduces photoreceptor cell stress,preventing photoreceptor cell decline in viability and death in variousdiseases of vision loss, and in normal age-related decline in dim-lightand peripheral rod-mediated vision, central cone-mediated vision, andloss of night vision.

In another aspect of the invention, the opsin-binding agent promotes thedegradation of the mislocalized opsin protein. This type ofopsin-binding agent is referred to as a “Counterproductive”, Shipwreck”,or “Destructive Chaperone.”

Enhancing the degradation of the mislocalized opsin by such an agentreduces the amount of mislocalized protein, thereby relievingphotoreceptor cell stress, preventing decline in viability and death ofphotoreceptor cells in diseases of vision loss, as well as in normalage-related decline in dim-light and peripheral rod-mediated vision,central cone-mediated vision, and loss of night vision.

In embodiments of the foregoing, the ocular protein mislocalizationdisorder is one or more of wet or dry form of macular degeneration,retinitis pigmentosa, a retinal or macular dystrophy, Stargardt'sdisease, Sorsby's dystrophy, autosomal dominant drusen, Best'sdystrophy, peripherin mutation associate with macular dystrophy,dominant form of Stargart's disease, North Carolina macular dystrophy,light toxicity, retinitis pigmentosa, normal vision loss related agingand normal loss of night vision related to aging.

Opsin, the GPCR (G-protein coupled receptor) responsible for vision,readily regenerates with 11-cis-retinal to form the visual pigmentrhodopsin. The pigment is generated by formation of a protonated Schiffbase between the aldehyde group of 11-cis-retinal and the ε-amino groupof L-lysine in opsin (Matsumoto and Yoshizawa, Nature 1975 Dec. 11;258(5535):523-6).

Thus, the present invention provides compositions and methods of use ofsmall molecule compounds that bind to wild type and mutant opsins andcompete with, or other wise prevent, 11-cis-retinal from combining withopsin to form rhodopsin and thereby inhibit formation of 11-cis-retinaland other visual cycle products.

In one embodiment, the invention provides opsin binding ligands ofFormula I and pharmaceutically acceptable salts thereof:

wherein R¹ and R² are independently:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) —CH₂CH₃;

R_(a) and R_(b) are each independently:

-   -   1) hydrogen,    -   2) —CH₃, or    -   3) —CH₂CH₃;

A

B

C

D

E is:

-   -   1) C(H)—C(═O)—C(R³)═C(R⁴)—C(R³),    -   2) C(H)—C(═N—OR⁵)C(R^(3′))═C(R⁴)—C(R³),    -   3) C(H)—C(H)(OR⁵)—C(R^(3′))═C(R⁴)—C(R³),    -   4) C(H)—C(═O)—C(H)(R^(3′))—C(H)(R⁴)—C(R³),    -   5) C(H)—C(═N—OR⁵)—C(H)(R³)—C(H)(R⁴)—C(R³),    -   6) C(H)—C(H)(OR⁵)—C(H)(R³)—C(H)(R⁴)—C(R³),    -   7) C(H)—CH₂—C(═O)—N(R⁴)—C(R³),    -   8) C(H)—CH₂—C(═O)—O—C(R³),    -   9) C(H)—C(H)(R^(3′))—C(H)(R⁴)—C(═O)—C(R³),    -   10) C(H)—CH₂—CH₂—C(═N—OR⁵)—C(R³),    -   11) C═C(R⁴)—C(═O)—C(R^(3′))(R⁴)—C(R³),    -   12) C═C(R⁴)—C(═N—OR⁵)—C(R^(3′))(R^(4′))—C(R³),    -   13) C═C(R⁴)—C(H)(OR⁵)—C(R^(3′))(R^(4′))—C(R³),    -   14) C(H)—CH₂—C(R⁶)═C(H)—C(R³),    -   15) C(H)—CH₂—C(R⁶)—C(H)(R⁷)—C(R³),    -   16) C(H)—C(═O)—C(R^(3′))(R^(4′))—C(R⁴)(R^(5′))—C(R³),    -   17) C(H)—C(H)(OR⁵)—C(R^(3′))(R^(4′))—C(R⁴)(R^(5′))—C(R³),    -   18) C(H)—C(═O)—N(R⁴)—CH₂—C(R³),    -   19) C(H)—C(R^(3′))(R⁵)—C(═O)—C(R⁴)═C,    -   20) C(H)—C(R^(3′))(R⁵)—C(═N—OR⁵)—C(R⁴)═C,    -   21) C(H)—C(R^(3′))(R⁵)—C(H)(OR⁵)—C(R⁴)═C,    -   22) C(H)—C(R^(3′))(R⁵)—C(═O)—C(R⁴)(R³)—C(R^(4′)),    -   23) C(H)—C(R^(3′))(R⁵)—C(═N—OR^(5′))—C(R⁴)(R³)—C(R^(4′)),    -   24) C(H)—C(R³)(R⁵)—C(H)(OR⁵)—C(R⁴)(R³)—C(R^(4′)),    -   25) /=C—C(═O)—N(R⁴)—C(R^(3′))(R⁵)—C=/,    -   26) C(H)—C(R^(3′))(R⁵)—C(R⁴)(R³)—C(H)(OR^(5′))—C(R^(4′)),    -   27) C(H)—C(═N—OR⁵)—C(R³)(R^(4′))—C(R⁴)(R⁵)—C(R³), or    -   28) /=C—C(R³)(R⁵)—C(R⁴)(R^(5′))—C(O)—C=/,    -   29) C(H)—C(R^(3′))═C(R⁴)—C(O)—C(R³), or    -   30) C(H)—C(R^(3′))═C(R⁴)—C(H)(OR⁵)—C(R³);

wherin /=preceding A and =/ following E are meant to denote a doublebond between A and E.

R³, R^(3′), R⁵ and R^(5′) are each independently:

-   -   1) hydrogen, or    -   2) lower alkyl;

R⁴ and R^(4′) are each independently:

-   -   1) hydrogen,    -   2) lower alkyl,    -   3) cycloalkyl, or    -   4) phenyl;

R⁶ is:

-   -   1) —CO₂CH₃,    -   2) —CONR⁵R^(5′), or    -   3) —CH²OR⁵;

R⁷ is:

-   -   1) hydrogen, or    -   2) —OR⁵;

with the proviso that if either R¹ or R² are selected as hydrogen thenR_(a) and R_(b) must be selected as methyl or ethyl.

In preferred embodiments, the compound has the structure of Formula Iwherein A

B

C

D

E is C═C(R⁴)—C(═O)—C(R^(3′))(R^(4′))—C(R³) and R³, R^(3′), R⁴ and R^(4′)are each independently hydrogen or lower alkyl or more preferably areeach independently hydrogen or methyl and most preferably R³, R^(3′), R⁴and R^(4′) are hydrogen. In other preferred embodiments, R¹ and R² areeach independently methyl or ethyl and R_(a) and R_(b) are eachindependently hydrogen or methyl or more preferably R¹ and R² are methyland R_(a) and R_(b) are each independently hydrogen or methyl, and mostpreferably R¹ and R² are methyl and R_(a) and R_(b) are hydrogen.

In preferred embodiments of compounds of Formula I, lower alkyl ismethyl or ethyl, most preferably methyl.

In other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C═C(R⁴)—C(═O)—C(R^(3′))(R^(4′))—C(R³) and R³, R^(3′), R⁴ and R^(4′)are each independently hydrogen or lower alkyl or more preferably areeach independently hydrogen or methyl, and most preferably R³, R^(3′)and R⁴ are hydrogen and R^(4′) is lower alkyl or methyl. In otherpreferred embodiments R¹ and R² are each independently methyl or ethyland R_(a) and R_(b) are each independently hydrogen or methyl or, morepreferably, R¹ and R² are methyl and R_(a) and R_(b) are eachindependently hydrogen or methyl, and most preferably R¹ and R² are bothmethyl and R_(a) and R_(b) are both hydrogen.

In other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C═C(R⁴)—C(═O)—C(R^(3′))(R^(4′))—C(R³) and R³, R^(3′), R⁴ and R^(4′)are each independently hydrogen or lower alkyl or more preferably areeach independently hydrogen or methyl, and most preferably wherein R³,R^(3′), R⁴ and R^(4′) are hydrogen. In other preferred embodiments, R¹and R² are methyl or ethyl and R_(a) and R_(b) are hydrogen or methyl orpreferably R¹ and R² are methyl and R_(a) and R_(b) are hydrogen ormethyl, and most preferably R¹, R², R_(a) and R_(b) are each methyl.

In other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C(H)—C(═O)—C(R³)═C(R⁴)—C(R³) and R³, R^(3′), R⁴ and are hydrogen orlower alkyl, or more preferably are each independently hydrogen ormethyl, or most preferably R³ is hydrogen and R^(3′) and R⁴ are methyl.In other preferred embodiments, R¹ and R² are each independentlyhydrogen or methyl and R_(a) and R_(b) are each independently methyl orethyl, or more preferably R¹ and R² are each independently hydrogen andR_(a) and R_(b) are each independently methyl or ethyl, and mostpreferably R¹ and R² are each hydrogen and R_(a) and R_(b) are eachmethyl.

In other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C(H)—C(R³)(R⁵)—C(H)(OR⁵)—C(R⁴)(R³)—C(R^(4′)) and R³, R^(3′), R⁴,R^(4′), R⁵ and R^(5′) are hydrogen or lower alkyl more preferablyhydrogen or methyl most preferably R³, R^(3′), R⁴, R⁵ and R^(5′) arehydrogen and R^(4′) is methyl. In other preferred examples, R¹ and R²are each independently methyl or ethyl and R_(a) and R_(b) are eachindependently hydrogen or methyl, or more preferably R¹ and R² are eachindependently methyl and R_(a) and R_(b) are each independently hydrogenor methyl, or most preferably R¹ and R² are methyl and R_(a) and R_(b)are hydrogen.

In other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C(H)—C(R^(3′))(R⁵)—C(═N—OR^(5′))—C(R⁴)(R³)—C(R^(4′)) and R³,R^(3′), R⁴, R^(4′), R⁵ and R^(5′) are hydrogen or lower alkyl, or morepreferably hydrogen or methyl, and most preferably R³, R^(3′), R⁴, R⁵and R^(5′) are hydrogen and R^(4′) is methyl. In other preferredembodiments, R¹ and R² are each independently methyl or ethyl and R_(a)and R_(b) are each independently hydrogen or methyl, or more preferablyR¹ and R² are methyl and R_(a) and R_(b) are each independently hydrogenor methyl, and most preferably R¹ and R² are methyl and R_(a) and R_(b)are hydrogen.

In other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C(H)—C(H)(OR⁵)—C(R³)═C(R⁴)—C(R³) and R³, R^(3′), R⁴ and R⁵ are eachindependently hydrogen or lower alkyl, or more preferably are eachindependently hydrogen or methyl, or most preferably R³, R^(3′), R⁴ aremethyl and R⁵ is hydrogen. In other preferred embodiments, R¹ and R² areeach independently methyl or ethyl and R_(a) and R_(b) are eachindependently hydrogen or methyl, or more preferably R¹ and R² aremethyl and R_(a) and R_(b) are each independently hydrogen or methyl, ormost preferably R¹ and R² are methyl and R_(a) and R_(b) are hydrogen.

In yet other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C(H)—C(H)(OR⁵)—C(H)(R³)—C(H)(R⁴)—C(R³) and R³, R^(3′), R⁴ and R⁵are each independently hydrogen or lower alkyl, or more preferably areeach independently hydrogen or methyl, and most preferably R^(3′), R⁴and R⁵ are hydrogen and R³ is methyl. In other preferred embodiments, R¹and R² are each independently methyl or ethyl and R_(a) and R_(b) areeach independently hydrogen or methyl, or more preferably R¹ and R² aremethyl and R_(a) and R_(b) are each independently hydrogen or methyl, ormost preferably R¹ and R² are methyl and R_(a) and R_(b) are hydrogen.

In yet other preferred embodiments, the compound has the structure ofFormula I wherein A

B

C

D

E is C(H)—C(R^(3′))(R⁵)—C(═O)—C(R⁴)(R³)—C(R^(4′)) and R³, R^(3′), R⁴,R^(4′) and R⁵ are each independently hydrogen or lower alkyl, or morepreferably are each independently hydrogen or methyl, and mostpreferably R³, R^(3′), R⁴, R^(4′) and R⁵ are hydrogen. In otherpreferred embodiments, R¹ and R² are each independently methyl or ethyland R_(a) and R_(b) are each independently hydrogen or methyl, or morepreferably R¹ and R² are methyl and R_(a) and R_(b) are eachindependently hydrogen or methyl, or most preferably R¹, R², R_(a) andR_(b) are methyl.

In preferred examples of the invention, the compound has the structureof Formula I wherein R¹ and R² are both methyl and R_(a) and R_(b) areboth hydrogen, or where R¹ and R² are both hydrogen and R_(a) and R_(b)are both methyl or where R¹, R², R_(a) and R_(b) are all methyl.

In another embodiment, the invention provides opsin binding ligands ofFormula (II) and pharmaceutically acceptable salts thereof:

wherein T is:

-   -   1) C(═O),    -   2) C(H)(OR⁵), or    -   3) C(═N—OR⁵);

R_(c) and R_(d) are each independently:

-   -   1) —CH₃, or    -   2) —CH₂CH₃;

and R⁴ is as defined herein.

In preferred embodiments, the compound has the structure of Formula IIwherein T is C(═O) or C(═N—OR⁵) and R⁴ is lower alkyl, and R_(c) andR_(d) are methyl or ethyl or more preferably wherein T is C(═O) andR_(c) and R_(d) are both methyl and most preferably wherein R⁴ ismethyl.

In specific embodiments the opsin binding compound of Formula I is(wherein each compound number corresponds to the number of the examplewhere it was prepared):

-   (±)-(3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 1);-   (±)-(3aS,7aR)-4,4-dimethylhexahydro-1H-indol-2(3H)-one (Compound 2);-   (±)-(3aS,7aS)-4,4,7,7-tetramethylhexahydrobenzofuran-2(3H)-one    (Compound 3);-   (±)-(3aS,7aR)-1,4,4-trimethylhexahydro-1H-indol-2(3H)-one (Compound    4);-   (±)-(3aS,7aR)-4,4,7a-trimethyloctahydro-1H-inden-1-one (Compound 5);-   4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one (Compound    6);-   4,4-dimethylhexahydrobenzofuran-2(3H)-one (Compound 7);-   (±)-(3aS,7aS)-methyl    3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxylate    (Compound 8);-   (±)-(3R,3aS,7aS)-3,3a,7,7-tetramethyloctahydro-1H-inden-1-one    (Compound 9);-   4,4,7a-trimethyl-2,4,5,6,7,7a-hexahydro-1H-inden-2-ol (Compound 10);-   2-methoxy-4,4,7a-trimethyl-2,4,5,6,7,7a-hexahydro-1H-indene    (Compound 11);-   7,7-dimethyl-2,3,4,5,6,7-hexahydro-1H-isoindol-1-one (Compound 12);-   2,7,7-trimethyl-2,3,4,5,6,7-hexahydro-1H-isoindol-1-one (Compound    13);-   (±)-(3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    oxime (Compound 14);-   (±)-(1S,3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol    (Compound 15);-   (±)-(3R,3aS,7aS)-3,3a,7,7-tetramethyloctahydro-1H-inden-1-one oxime    (Compound 16);-   7,7-dimethyloctahydro-1H-isoindol-1-one (Compound 17);-   (±)-(1S,3aS,7aR)-4,4,7a-trimethyloctahydro-1H-inden-1-ol (Compound    18);-   (±)-(3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    O-methyl oxime (Compound 19);-   (±)-(1R,7aS)-1,4,4,7a-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 20);-   1,1,4,4,7a-pentamethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 21);-   (±)-((3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahyrdo-1H-inden-2-yl)methanol    (Compound 22);-   (±)-(3aS,7aS)-2-(methoxymethyl)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene    (Compound 23);-   (±)-(1S,3aS,7aR)-1-methoxy-4,4,7a-trimethyloctahydro-1H-indene    (Compound 24);-   (±)-(3aS,7aS)-methyl    3a,7,7-trimethyloctahydro-1H-indene-2-carboxylate (Compound 25);-   (±)-((3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-2-yl)methanol    (Compound 26);-   7,7-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one (Compound 27);-   (±)-(3aR,7aS)-3a,7,7-trimethyl-3-phenyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 28);-   (±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 29);-   4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one oxime    (Compound 30);-   4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one O-methyl    oxime (Compound 31);-   (±)-(3R,3aS,7aS)-3,3a,7,7-tetramethyloctahydro-1H-inden-1-ol    (Compound 32);-   (±)-(3aS,7aR)-4,4,7a-trimethyloctahydro-1H-inden-1-one oxime    (Compound 33);-   (±)-(3aR,7aS)-3-ethyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 34);-   (±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 35);-   (±)-(1S,3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol    (Compound 36);-   (±)-(1R,3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol    (Compound 37);-   (±)-(1S,2R,3aS,7aR)-2-(hydroxymethyl)-4,4,7a-trimethyloctahydro-1H-inden-1-ol    (Compound 38);-   (±)-(1S,2S,3aS,7aR)-2-(hydroxymethyl)-4,4,7a-trimethyloctahydro-1H-inden-1-ol    (Compound 39);-   (±)-(3aS,7aS)-3a,7,7-trimethylhexahydro-1H-inden-2(3H)-one (Compound    40);-   4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one (Compound    41);-   (±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxamide    (Compound 42);-   (±)-(3aR,7aS)-3-tert-butyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 43);-   (±)-(3aR,7aS)-3-cyclopropyl-3a,7,7-trimethyl-3a,4,    5,6,7,7a-hexahydro-1H-inden-1-one (Compound 44);-   (±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    oxime (Compound 45);-   (±)-(3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-one    (Compound 46);-   (±)-(1S,3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol    (Compound 47);-   (±)-(3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-one    O-methyl oxime (Compound 48);-   (±)-(3aS,7aS)-3a,7,7-trimethyloctahydro-1H-indene-2-carboxamide    (Compound 49);-   (±)-(1R,3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-ol    (Compound 50);-   (±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    O-methyl oxime (Compound 51);-   (±)-(3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-one    oxime (Compound 52);-   4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one (Compound 53);-   4,4-dimethylhexahydro-1H-inden-2(3H)-one (Compound 54);-   4,4-dimethylhexahydro-1H-inden-2(3H)-one oxime (Compound 55);-   4,4-dimethyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one (Compound 56);-   (±)-(3aS,7aS)-2-(methoxymethyl)-3a,7,7-trimethyloctahydro-1H-indene    (Compound 57);-   (±)-(1S,3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-ol    (Compound 58);-   (±)-(3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-1-one (Compound    59);-   (±)-(1S,3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-1-ol (Compound    60);-   (±)-(1R,7aS)-1-isopropyl-4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 61);-   (±)-(3aS,7aS)-3a,7,7-trimethylhexahydro-1H-inden-2(3H)-one oxime    (Compound 62);-   (±)-(3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-2-ol (Compound 63);-   (±)-(3aS,7aS)-2,3,4,4-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 64);-   (±)-(3aR,7aS)-2,3,3a,7,7-pentamethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one    (Compound 65);-   (±)-(1R,3aR,7aS)-2,3,3a,7,7-pentamethyl-3a,4,5,6,7,7a-hexahydro 1H    inden-1-ol (Compound 66);-   (±)-(1R,7aS)-1,4,4-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 67);-   (±)-(1R,3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-1-ol (Compound    68);-   (+)-4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 69a);-   (−)-4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 69b);-   (3aR,7aS) and    (3aS,7aR)-4,4,7,7-tetramethylhexahydro-1H-inden-2(3H)-one (Compound    70);-   (±)-(3R,3aR)-3,4,4-trimethyl-2,3,3a,4,5,6-hexahydro-1H-inden-1-one    (Compound 71);-   (±)-(1S,3aS,7aS)-2,3,4,4-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol    (Compound 72);-   (±)-(3aS,7aS)-2,3,4,4-tetramethyloctahydro-1H-inden-1-one (Compound    73);-   (±)-(3R,3aS,7aR)-3,7,7-trimethyloctahydro-1H-inden-1-one (Compound    74);-   7a-ethyl-4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 75);-   (±)-(1R,7aS)-1-ethyl-4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one    (Compound 76);-   (±)-(3aR,7aS)-4,4,7,7-tetramethyloctahydro-1H-inden-2-ol (Compound    77);-   4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one oxime    (Compound 78);-   (±)-(3aR,7aS)-4,4,7,7-tetramethylhexahydro-1H-inden-2(3H)-one oxime    (Compound 79)    including all pharmaceutically acceptable salts, hydrates, or    solvates thereof.

All compound names were derived using ChemBioDraw 11.0.1.

The present invention does not include the following compounds (asrepresented by their indicated CAS registry numbers) as novelcompositions of matter (but are claimed for use in the methods of theinvention) and are referred to herein as the Excluded Compound Group:1322705-86-1, 1217498-30-0, 917392-28-0, 901132-06-7, 678160-04-8,531512-99-9, 531512-94-4, 531512-91-1, 531512-87-5, 531512-85-3,531512-83-1, 531512-76-2, 334826-84-5, 226546-70-9, 170081-07-9,165402-62-0, 108613-26-9, 101098-92-4, 75824-85-0, 75824-83-8,75824-82-7, 75824-78-1, 70006-17-6, 55085-49-9, 42741-51-5, 38881-23-1,35076-54-1, 31089-96-0, 31089-92-6, 28102-31-0, 28102-26-3, 28102-23-0,1258783-39-9, 155501-25-0, 104641-34-1, 104640-83-7, 104527-56-2,87220-77-7, 71075-17-7, 38725-47-2, 37531-06-9, 24739-75-1, 16778-27-1,136771-91-0, 136771-90-9 and 369366-32-5.

The Excluded Compound Group contains any and all of the compounds in thepreceding list as identified by their indicated CAS (Chemical AbstractsService) numbers.

Thus, the novel compounds and/or compositions of matter of the inventionare compounds of Formula I (including their indicated substituentidentities) but do not include compounds of the Excluded Compound Group.

However, the methods of the present invention employ any compounds ofFormula I (including their indicated substituent identities) but do notexclude use of compounds of the Excluded Compound Group.

Especially preferred examples of the compounds of the invention, andmethods using said compounds, include compounds selected from one ormore of the group consisting of compounds 8, 13, 14, 16, 20, 22, 26, 27,29, 30, 33, 34, 36, 37, 41, 44, 45, 46, 53, 55, 58, 60, 62, 63, 64, 66,67, 69a, 70, 71 and 72 including all pharmaceutically acceptable salts,solvates and hydrates thereof.

Another embodiment of the invention provides the opsin binding ligandmetabolites of the opsin binding compounds. These metabolites, includebut are not limited to, degradation products, hydrolysis products,gluconoride adducts and the like, of the opsin binding compounds andpharmaceutically acceptable salts thereof, of the opsin compounds.

Another embodiment of the invention provides processes for making thenovel compounds of the invention and to the intermediates useful in suchprocesses. The reactions are performed in solvents appropriate to thereagents and materials used are suitable for the transformations beingeffected. It is understood by one skilled in the art of organicsynthesis that the functionality present in the molecule must beconsistent with the chemical transformation proposed. This will, onoccasion, necessitate judgment by the routineer as to the order ofsynthetic steps, protecting groups required, and deprotectionconditions. Substituents on the starting materials may be incompatiblewith some of the reaction conditions required in some of the methodsdescribed, but alternative methods and substituents compatible with thereaction conditions will be readily apparent to one skilled in the art.The use of sulfur, nitrogen and oxygen protecting groups is well knownfor protecting thiol, amino and alcohol groups against undesirablereactions during a synthetic procedure and many such protecting groupsare known and described by, for example, Greene and Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, John Wiley & Sons, New York(1999).

Compounds of the invention that have one or more asymmetric carbon atomsmay exist as the optically pure enantiomers, pure diastereomers,mixtures of enantiomers, mixtures of diastereomers, racemic mixtures ofenantiomers, diasteromeric racemates or mixtures of diastereomericracemates. It is to be understood that the invention anticipates andincludes within its scope all such isomers and mixtures thereof.

The chemical reactions described herein are generally disclosed in termsof their broadest application to the preparation of the compounds ofthis invention. Occasionally, the reactions may not be applicable asdescribed to each compound included within the disclosed scope. Thecompounds for which this occurs will be readily recognized by oneskilled in the art. In all such cases, either the reactions can besuccessfully performed by conventional modifications known to oneskilled in the art, e.g., by appropriate protection of interferinggroups, by changing to alternative conventional reagents, by routinemodification of reaction conditions, or other reactions disclosed hereinor otherwise conventional, will be applicable to the preparation of thecorresponding compounds of this invention. In all preparative methods,all starting materials are known or readily prepared from known startingmaterials.

Methods of the Invention

The present invention provides a method of using compounds of theFormula I for reducing the formation of toxic visual cycle products,comprising contacting an opsin protein with small molecule ligands thatreversibly bind to said opsin protein to inhibit 11-cis-retinal bindingin said binding pocket, thereby reducing formation of toxic visual cycleproducts associated with wet or dry ARMD. and reducing photocellapoptosis associated with excessive rhodopsin activation as a result ofbright light stimulation.

The present invention also provides a method of use of compounds of theFormula I for treating, preventing or reducing the risk of lighttoxicity in a mammal, comprising administering to a mammal, at risk ofdeveloping an ophthalmic condition that is related to the formation oraccumulation of a visual cycle product or apoptotic photocell death.

The present invention also provides a method of use of compounds of theFormula I for treating, preventing or reducing the risk of lighttoxicity in a mammal, comprising administering to a mammal, at risk ofdeveloping an ophthalmic condition that is related to the formation oraccumulation of a visual cycle product or apoptotic photocell death, aneffective amount of a that small molecule ligand that reversibly binds(for example, at or near the retinal binding pocket) to an opsin proteinpresent in the eye of said mammal, for example, to inhibit11-cis-retinal binding in said binding pocket, thereby reducing lighttoxicity and photocell apoptosis.

The present invention also provides a method of use of compounds of theFormula I for treating, preventing or reducing the risk of RP in amammal, comprising administering to a mammal, at risk of RP related tothe improper folding and trafficking of mutant opsins, an effectiveamount of a that small molecule ligand that reversibly binds (forexample, at or near the retinal binding pocket) to an opsin proteinpresent in the eye of said mammal, for example, to inhibit11-cis-retinal binding in said binding pocket, thereby reducing thevision loss caused by RP.

In specific examples of such methods, the small molecule ligand isselective for binding to opsin and/or the small molecule ligand binds tosaid opsin in the retinal binding pocket of said opsin protein and/orthe small molecule ligand binds to said opsin protein so as to inhibitcovalent binding of 11-cis-retinal to said opsin protein when said11-cis-retinal is contacted with said opsin protein when said smallmolecule ligand is present and/or the mammal is a human being.

In one embodiment, light toxicity is related to an ophthalmic procedure,for example, ophthalmic surgery. Said agent may be administered priorto, during or after said surgery (or at any one or more of those times).

In specific embodiments of the methods of the invention, the nativeopsin protein is present in a cell, such as a rod cell, preferably, amammalian and more preferably a human cell. In specific embodiments, thesmall molecule ligands of the invention inhibit binding of11-cis-retinal in the binding pocket of opsin and slow the visual cyclethereby reducing the formation of all-trans-retinal, or a toxic visualcycle product formed from it, such as lipofuscin orN-retinylidene-N-retinylethanolamine (A2E). Alternatively, photocellapoptosis as a result of excessive rhodopsin activation is reduced orprevented by inhibition of rhodopsin formation. Additionally, improperfolding and trafficking of mutant opsin proteins associated with RP isreduced.

In methods of the invention, administering is preferably by topicaladministration (such as with an eye wash) or by systemic administration(including oral, intraocular injection or periocular injection). By wayof preferred example, the ophthalmic condition to be treated is lighttoxicity, such as that resulting from ocular surgery, for example,retinal or cataract surgery.

Also encompassed is an ophthalmologic composition comprising aneffective amount of compounds of the Formula I in a pharmaceuticallyacceptable carrier, wherein said agent reversibly binds non-covalently(for example, at or near the retinal binding pocket) to said opsinprotein to inhibit 11-cis-retinal binding in said pocket, preferablywhere the small molecule ligand is selective for opsin protein.

The present invention further provides a screening method foridentifying a small molecule ligand that reduces light toxicity in amammalian eye, comprising:

(a) contacting a native opsin-protein with a test compound in thepresence of 11-cis-retinal and under conditions that promote the bindingof the test compound and the 11-cis-retinal to the native opsin protein;and

(b) determining a reversible reduction in rate of formation of rhodopsinrelative to the rate when said test compound is not present, therebyidentifying said test compound as a small molecule ligand that reduceslight toxicity in a mammalian eye. In a preferred embodiment, said testcompound is structurally related to a compound disclosed herein.

The compounds of the Formula I may be administered along with otheragents, including a mineral supplement, an anti-inflammatory agent, suchas a steroid, for example, a corticosteroid, and/or an anti-oxidant.Among the corticosteroids useful for such administration are thoseselected from the group consisting of cortisone, hydrocortisone,prednisone, prednisolone, methylprednisolone, triamcinolone,betamethasone, beclamethasone and dexamethasone. Useful anti-oxidantsinclude vitamin A, vitamin C and vitamin E.

The methods of the invention also contemplate reducing light toxicity byusing at least one additional agent (in addition to the compounds of theFormula I selected from the group consisting of a proteasomal inhibitor,an autophagy inhibitor, a lysosomal inhibitor, an inhibitor of proteintransport from the ER to the Golgi, an Hsp90 chaperone inhibitor, a heatshock response activator, a glycosidase inhibitor, and a histonedeacetylase inhibitor, wherein the small molecule opsin binding and theadditional compound are administered simultaneously or within fourteendays of each other in amounts sufficient to treat the subject.

In a particular example of the methods of the invention, the compoundsof the Formula I and the additional compound are administered within tendays of each other, within five days of each other, within twenty-fourhours of each other and preferably are administered simultaneously. Inone example, the small molecule opsin binding and the additionalcompound are administered directly to the eye. Such administration maybe intraocular or intravitrial. In other examples, the small moleculeopsin binding and the additional compound are each incorporated into acomposition that provides for their long-term release, such as where thecomposition is part of a microsphere, nanosphere, nano emulsion orimplant.

As described herein, the compounds of the Formula I are useful in themethods of the invention are available for use alone or in combinationwith one or more additional compounds to treat or prevent conditionsassociated with excessive rhodopsin activation, such as light toxicity,for example, resulting from ocular surgical procedures. In oneembodiment, compounds of the Formula I of the invention is administeredwithout an additional active compound. In another embodiment, compoundsof the Formula I of the invention is used in combination and withanother active compound (e.g., as discussed herein). In still anotherexemplary embodiment, compounds of the Formula I are administered incombination with the proteasomal inhibitor MG132, the autophagyinhibitor 3-methyladenine, a lysosomal inhibitor ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce formation of visual cycle products and cell apoptosis as aresult of excessive rhodopsin activation.

As described herein, the compounds of the Formula I are useful in themethods of the invention are available for use alone or in combinationwith one or more additional compounds to treat or prevent the aberrantprocessing and trafficking of mutant opsin proteins associated with rodcell death as a result of RP. In one embodiment, compounds of theFormula I of the invention is administered without an additional activecompound. In another embodiment, compounds of the Formula I of theinvention is used in combination and with another active compound (e.g.,as discussed herein). In still another exemplary embodiment, compoundsof the Formula I are administered in combination with the proteasomalinhibitor MG132, the autophagy inhibitor 3-methyladenine, a lysosomalinhibitor ammonium chloride, the ER-Golgi transport inhibitor brefeldin.A, the Hsp90 chaperone inhibitor Geldamycin, the heat shock responseactivator Celastrol, the glycosidase inhibitor, and the histonedeacetylase inhibitor Scriptaid, can be used to reduce or prevent therod cell death and resulting blindness associated with RP.

As described herein, the compounds of the Formula I are useful in themethods of the invention are available for use alone or in combinationwith one or more additional compounds to treat or prevent conditionsassociated with production and accumulation of toxic visual cycleproducts derived from all-trans-retinal, such as lipofucin and A2E, forexample, the blindness associated with wet or dry ARMD. In oneembodiment, compounds of the Formula I of the invention are administeredwithout an additional active compound. In another embodiment, compoundsof the Formula I of the invention are used in combination and withanother active compound (e.g., as discussed herein). In still anotherexemplary embodiment, compounds of the Formula I are administered incombination with the proteasomal inhibitor MG132, the autophagyinhibitor 3-methyladenine, a lysosomal inhibitor ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce formation of toxic visual cycle product metabolites and photocell death as a result of dry ARMD.

In a typical competition assay of the invention, a compound is soughtthat will tie up the retinal binding pocket of the opsin protein. Thus,the assay seeks to identify a small molecule opsin binding compound (onethat will not be tightly regulated by the retina as to amount enteringrod cells) that competes with or prevents 11-cis-retinal or9-cis-retinal from forming rhodopsin or isorhodopsin. Over time, thiswill slow the rate of formation of rhodopsin relative to the rate when11-cis-retinal alone is present. In one embodiment, the assay isconducted in the presence of 11-cis-retinal, and the rate of formationof rhodopsin is measured as a way of determining competition for theretinal binding pocket, for example, by determining the rate of increasein the 500 nm peak characteristic for rhodopsin. No antibodies forrhodopsin are required for this assay. A useful compound will exhibit arate of rhodopsin formation that is at least about 2 to 5 fold lowerthan that observed in the presence of 11-cis-retinal when said testcompound is not present.

In specific embodiments of the methods of the invention, the mis-foldedopsin protein comprises a mutation in its amino acid sequence, forexample, one of the mutations T17M, P347S, R135W or P23H, preferablyP23H.

Preferably, in any of the methods of the invention, the opsin-bindingagent binds to opsin in its retinal binding pocket.

In one aspect, the present invention provides a method of inhibiting theformation or accumulation of a visual cycle product, comprisingcontacting an opsin protein with a compound that reduces hydration ofsaid opsin protein, preferably wherein said compound competes with oneor more water molecules for binding to opsin. In specific embodiments ofsuch methods, the compound binds chemically to the opsin protein, forexample, through hydrogen bonding.

While use of any of the compounds disclosed herein as a means ofreducing hydration in the opsin binding pocket should be considered apreferred embodiment of such method, the reduction of formation of avisual cycle product by reducing the formation of rhodopsin is a generalmethod of the invention for reducing such visual cycle productformation, especially production of lipofuscin and/or A2E, and fortreating an ophthalmic disease by reducing said hydration is a generalaim of the invention and is not necessarily limited in scope only to theuse of chemicals disclosed herein but may include use of other known oryet to be known chemical compounds so long as they function in themethods of the invention and reduce hydration (i.e., binding of water)in the retinal binding pocket of opsin.

It should be noted that the compounds disclosed herein for use in themethods of the invention may not function to reduce hydration in theretinal binding pocket of opsin but may still function in one or more ofthe methods of the invention. For example, a compound of Formula I maybind to an allosteric site on the protein thereby excluding retinal fromthe retinal binding site without necessarily decreasing hydration yetstill reduce formation of a visual cycle product, such as lipofuscinand/or A2E, by virtue of its excluding retinal from the binding pocket,thus non-covalently reducing the activity of the visual cycle.

In embodiments of any of the compositions and methods of the invention,the opsin-binding agent (e.g., a non-retinoid binding agent) isselective for binding to opsin. Such selectivity is not to be taken asrequiring exclusivity that said agent may bind to other proteins as wellas to opsin but its binding to opsin will be at least selective, wherebythe binding constant (or dissociation constant) for binding to opsinwill be lower than the average value for binding to other proteins thatalso bind retinoids, such as retinal analogs. Preferably, opsin bindingagents are non-retinoid opsin-binding agents that bind non-covalently toopsin. Preferably, the opsin binding agent binds at or near the opsinretinal binding pocket, where the native ligand, 11-cis-retinal,normally binds. Without wishing to be bound by theory, in one embodimentthe binding pocket accommodates retinal or an agent of the invention,but not both. Accordingly, when an agent of the invention is bound at ornear the retinal binding pocket, other retinoids, such as11-cis-retinal, are unable to bind to opsin. Binding of an agent of theinvention inside the retinal binding pocket of a mis-folded opsinmolecule serves to direct formation of the native or wild-typeconformation of the opsin molecule or to stabilize a correctly foldedopsin protein, thereby facilitating insertion of the nowcorrectly-folded opsin into the membrane of a rod cell. Again, withoutwishing to be bound by theory, said insertion may help to maintain thewild-type conformation of opsin and the opsin-binding agent is free todiffuse out of the binding pocket, whereupon the pocket is available forbinding to retinal to form light-sensitive rhodopsin.

Other methods of the invention provide a means to restore photoreceptorfunction in a mammalian eye containing a mis-folded opsin protein thatcauses reduced photoreceptor function, comprising contacting saidmis-folded opsin protein with an opsin-binding agent (e.g., anon-retinoid) that reversibly binds (e.g., that binds non-covalently) ator near the retinal binding pocket. In other embodiments, binding of theopsin-binding agent to the mis-folded opsin protein competes with11-cis-retinal for binding in said binding pocket. Desirably, binding ofthe opsin-binding agent restores the native conformation of saidmis-folded opsin protein.

In preferred embodiments, the mammalian eye is a human eye. Inadditional embodiments, said contacting occurs by administering saidopsin-binding agent (e.g., non-retinoid) to a mammal afflicted with anophthalmic condition, such as a condition characterized by reducedphotoreceptor function. In various embodiments, the condition is the wetor dry form of macular degeneration, diabetic RP, a retinal or maculardystrophy, Stargardt's disease, Sorsby's dystrophy, autosomal dominantdrusen, Best's dystrophy, peripherin mutation associate with maculardystrophy, dominant form of Stargart's disease, North Carolina maculardystrophy, light toxicity (e.g., due to retinal surgery), or retinitispigmentosa. The administration may be topical administration or bysystemic administration, the latter including oral administration,intraocular injection or periocular injection. Topical administrationcan include, for example, eye drops containing an effective amount of anagent of the invention in a suitable pharmaceutical carrier.

In another embodiment, the present invention also provides a method ofstabilizing a mutant opsin protein, comprising contacting said mutantopsin protein with a non-retinoid opsin-binding agent that reversiblybinds non-covalently (for example, at or in the retinal binding pocket)to said mutant opsin protein to prevent retinoid binding in said bindingpocket, thereby stabilizing said mutant opsin protein.

The present invention also provides a method of ameliorating loss ofphotoreceptor function in a mammalian eye, comprising administering aneffective amount of an opsin-binding agent, such as a non-retinoid, to amammal afflicted with a mutant opsin protein that has reduced affinityfor 11-cis-retinal, whereby the opsin binding agent reversibly binds(e.g., non-covalently) to the retinal binding pocket of said mutantopsin, thereby ameliorating loss of photoreceptor function in saidmammalian eye. In one embodiment, the contacting occurs by administeringsaid opsin-binding agent to a mammal afflicted with said reducedphotoreceptor function, wherein said administering may be by topicaladministration or by systemic administration, the latter including oral,intraocular injection or periocular injection, and the former includingthe use of eye drops containing an agent of the invention. Such loss ofphotoreceptor function may be a partial loss or a complete loss, andwhere a partial loss it may be to any degree between 1% loss and 99%loss. In addition, such loss may be due to the presence of a mutationthat causes mis-folding of the opsin, such as where the mutation is theP23H mutation. In another embodiment, the opsin binding agent isadministered to ameliorate an opthalmic condition related to themislocalization of an opsin protein. In one embodiment, the inventionprovides for the treatment of a subject having the dry form ofage-related macular degeneration, where at least a portion of the opsinpresent in an ocular photoreceptor cell (e.g., a rod or cone cell) ismislocalized. The mislocalized protein fails to be inserted into themembrane of a photoreceptor cell, where its function is required forvision. Administration of the opsin binding agent to a subject having amislocalized opsin protein rescues, at least in part, opsinlocalization. Accordingly, the invention is useful to prevent or treatan ophthalmic condition related to opsin mislocalization or toameliorate a symptom thereof.

The present invention provides a method for treating and/or preventingan ophthalmic condition or a symptom thereof, including but not limitedto, wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity (e.g., due to retinalsurgery), or retinitis pigmentosa in a subject, such as a human patient,comprising administering to a subject afflicted with, or at risk ofdeveloping, one of the aforementioned conditions or another ophthalmiccondition related to the expression of a misfolded or mislocalized opsinprotein using a therapeutically effective amount of an opsin-bindingagent, e.g., an agent that shows positive activity when tested in anyone or more of the screening assays of the invention.

Such a method may also comprise administering to said subject at leastone additional agent selected from the group consisting of a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp90 chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, anda histone deacetylase inhibitor, wherein the opsin-binding compound andthe additional compound are administered simultaneously or withinfourteen days of each other in amounts sufficient to treat the subject.

Here again the patient may comprise a mutation that affects proteinfolding where said mutation(s) causes mis-folding, e.g., in an opsinprotein, and may be any of the mutations recited elsewhere herein, suchas a P23H mutation. In other embodiments, the patient has an ophthalmiccondition that is related to the mislocalization of an opsin protein.The mislocalized opsin fails to insert into the membrane of aphotoreceptor cell (e.g., a rod or cone cell). In general, this failurein localization would effect only a portion of the opsin present in anocular cell of a patient.

In particular examples of the methods of the invention, theopsin-binding compound and the additional compound are administeredwithin ten days of each other, more preferably within five days of eachother, even more preferably within twenty-four hours of each other andmost preferably are administered simultaneously. In one example, theopsin-binding compound and the additional compound are administereddirectly to the eye. Such administration may be intra-ocular. In otherexamples, the opsin-binding compound and the additional compound areeach incorporated into a composition that provides for their long-termrelease, such as where the composition is part of a microsphere,nanosphere, or nano emulsion. In one example, the composition isadministered via a drug-delivery device that effects long-term release.Such methods also contemplate administering a vitamin A supplement alongwith an agent of the invention.

As described herein, the opsin-binding agents useful in the methods ofthe invention are available for use alone or in combination with one ormore additional compounds to treat or prevent conditions associated withthe wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity (e.g., due to retinalsurgery), retinitis pigmentosa or another ophthalmic condition relatedto the expression of a misfolded or mislocalized opsin protein. In oneembodiment, an opsin-hinding compound of the invention (e.g., anon-retinoid or a retinoid that fails to covalently bind to opsin) isadministered to a subject identified as having or at risk of developingsuch a condition. Optionally, the opsin binding agent is administeredtogether with another therapeutic agent. In another embodiment, anon-retinoid opsin-binding compound of the invention is used incombination with a synthetic retinoid (e.g., as disclosed in U.S. PatentPublication No. 2004-0242704), and optionally with another activecompound (e.g., as discussed herein). In still another exemplaryembodiment, an opsin-binding compound is administered in combinationwith the proteasomal inhibitor MG132, the autophagy inhibitor3-methyladenine, a lysosomal inhibitor, such as ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and/or the histone deacetylase inhibitor Scriptaid, or anyother agent that can stabilize a mutant P23H opsin protein in abiochemically functional conformation that allows it to associate with11-cis-retinal to form rhodopsin.

In specific embodiments, an opsin-binding compound is a non-polymeric(e.g., a small molecule, such as those disclosed herein for use in themethods of the invention) compound having a molecular weight less thanabout 1000 daltons, less than 800, less than 600, less than 500, lessthan 400, or less than about 300 daltons. In certain embodiments, acompound of the invention increases the amount (e.g., from or in a cell)of a stably-folded and/or complexed mutant protein by at least 10%, 15%,20%, 25%, 50%, 75%, or 100% compared to an untreated control cell orprotein.

Proteasomal Inhibitors

The 26S proteasome is a multicatalytic protease that cleaves ubiquinatedproteins into short peptides. MG-132 is one proteasomal inhibitor thatmay be used. MG-132 is particularly useful for the treatment of lighttoxicity and other ocular diseases related to the accumulation of visualcycle products (e.g., all-trans-retinal, A2E, lipofuscin), proteinaggregation or protein misfolding. Other proteasomal inhibitors usefulin combination with of the invention in the methods of the inventioninclude lactocystin (LC), clasto-lactocystin-beta-lactone, PSI(N-carbobenzoyl-Ile-Glu-(OtBu)-Ala-Leu-CHO), MG-132(N-carbobenzoyl-Leu-Leu-Leu-CHO), MG-115(N-carbobenzoyl-Leu-Leu-Nva-CHO), MG-101 (N-Acetyl-Leu-Leu-norLeu-CHO),ALLM (N-Acetyl-Leu-Leu-Met-CHO), N-carbobenzoyl-Gly-Pro-Phe-leu-CHO,N-carbobenzoyl-Gly-Pro-Ala-Phe-CHO, N-carbobenzoyl-Leu-Leu-Phe-CHO, andsalts or analogs thereof. Other proteasomal inhibitors and their usesare described in U.S. Pat. No. 6,492,333.

Autophagy Inhibitors

Autophagy is an evolutionarily conserved mechanism for the degradationof cellular components in the cytoplasm, and serves as a cell survivalmechanism in starving cells. During autophagy pieces of cytoplasm becomeencapsulated by cellular membranes, forming autophagic vacuoles thateventually fuse with lysosomes to have their contents degraded.Autophagy inhibitors may be used in combination with an opsin-binding oropsin-stabilizing compound of the invention. Autophagy inhibitors usefulin combination with a of the invention in the methods of the inventioninclude, but are not limited to, 3-methyladenine, 3-methyl adenosine,adenosine, okadaic acid, N⁶-mercaptopurine riboside (N⁶-MPR), anaminothiolated adenosine analog, 5-amino-4-imidazole carboxamideriboside (AICAR), bafilomycin A1, and salts or analogs thereof.

Lysosomal Inhibitors

The lysosome is a major site of cellular protein degradation.Degradation of proteins entering the cell by receptor-mediatedendocytosis or by pinocytosis, and of plasma membrane proteins takesplace in lysosomes. Lysosomal inhibitors, such as ammonium chloride,leupeptin, trans-epoxysaccinyl-L-leucylamide-(4-guanidino) butane,L-methionine methyl ester, ammonium chloride, methylamine, chloroquine,and salts or analogs thereof, are useful in combination with anopsin-binding or opsin-stabilizing compound of the invention.

HSP90 Chaperone Inhibitors

Heat shock protein 90 (Hsp90) is responsible for chaperoning proteinsinvolved in cell signaling, proliferation and survival, and is essentialfor the conformational stability and function of a number of proteins.HSP-90 inhibitors are useful in combination with an opsin-binding oropsin-stabilizing compound in the methods of the invention. HSP-90inhibitors include benzoquinone ansamycin antibiotics, such asgeldanamycin and 17-allylamino-17-demethoxygeldanamycin (17-AAG), whichspecifically bind to Hsp90, alter its function, and promote theproteolytic degradation of substrate proteins. Other HSP-90 inhibitorsinclude, but are not limited to, radicicol, novobiocin, and any Hsp90inhibitor that binds to the Hsp90 ATP/ADP pocket.

Heat Shock Response Activators

Celastrol, a quinone methide triterpene, activates the human heat shockresponse. In combination with an opsin-binding or opsin-stabilizingcompound in methods of the invention, celastrol and other heat shockresponse activators are useful for the treatment of PCD. Heat shockresponse activators include, but are not limited to, celastrol,celastrol methyl ester, dihydrocelastrol diacetate, celastrol butylester, dihydrocelastrol, and salts or analogs thereof.

Histone Deacetylase Inhibitors

Regulation of gene expression is mediated by several mechanisms,including the post-translational modifications of histones by dynamicacetylation and deacetylation. The enzymes responsible for reversibleacetylationl/deacetylation processes are histone acetyltransferases(HATs) and histone deacetylases (HDACs), respectively. Histonedeacetylase inhibitors include Scriptaid, APHA Compound 8, Apicidin,sodium butyrate, (−)-Depudecin, Sirtinol, trichostatin A, and salts oranalogs thereof. Such inhibitors may be used in combination withcompounds of the invention in the methods disclosed herein.

Glycosidase Inhibitors

Glycosidase inhibitors are one class of compounds that are useful in themethods of the invention, when administered in combination with anopsin-binding or opsin-stabilizing compound of the invention.Castanospermine, a polyhydroxy alkaloid isolated from plant sources,inhibits enzymatic glycoside hydrolysis. Castanospermine and itsderivatives are particularly useful for the treatment of light toxicityor of an ocular Protein Conformation Disorder, such as RP. Also usefulin the methods of the invention are other glycosidase inhibitors,including australine hydrochloride,6-Acetamido-6-deoxy-castanosperrnine, which is a powerful inhibitor ofhexosaminidases, Deoxyfuconojirimycin hydrochloride (DFJ7),Deoxynojirimycin (DNJ), which inhibits glucosidase I and II,Deoxygalactonojirimycin hydrochloride (DGJ), winch inhibitsα-D-galactosidase, Deoxymannojirimycin hydrochloride (DM1),2R,5R-Bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP), also knownas 2,5-dideoxy-2,5-imino-D-mannitol, 1,4-Dideoxy-1,4-imino-D-mannitolhydrochloride, (3R,4R,5R,6R)-3,4,5,6-Tetrahydroxyazepane Hydrochloride,which inhibits β-N-acetylglucosaminidase, 1,5-Dideoxy-1,5-imino-xylitol,which inhibits β-glucosidase, and Kifunensine, an inhibitor ofmannosidase 1. Also useful in combination with an opsin-binding oropsin-stabilizing compound are N-butyldeoxynojirimycin (EDNJ), N-nonylDNJ (NDND, N-hexyl DNJ (I5TDNJ), N-methyldeoxynojirimycin (MDNJ), andother glycosidase inhibitors known in the art. Glycosidase inhibitorsare available commercially, for example, from Industrial ResearchLimited (Wellington, New Zealand) and methods of using them aredescribed, for example, in U.S. Pat. Nos. 4,894,388, 5,043,273,5,103,008, 5,844,102, and 6,831,176; and in U.S. Patent Publication Nos.20020006909.

Pharmaceutical Compositions

The present invention features pharmaceutical preparations comprisingcompounds together with pharmaceutically acceptable carriers, where thecompounds provide for the inhibition of visual cycle products, such asall-trans-retinal or other products formed from 11-cis-retinal. Suchpreparations have both therapeutic and prophylactic applications. In oneembodiment, a pharmaceutical composition includes an opsin-binding orstabilizing compound (e.g., a compound identified using the methods ofExample 1) or a pharmaceutically acceptable salt thereof; optionally incombination with at least one additional compound that is a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp9O chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, ora histone deacetylase inhibitor. The opsin-binding or opsin-stabilizingcompound is preferably not a natural or synthetic retinoid. Theopsin-binding or opsin-stabilizing compound and the additional compoundare formulated together or separately. Compounds of the invention may beadministered as part of a pharmaceutical composition. The non-oralcompositions should be sterile and contain a therapeutically effectiveamount of the opsin-binding or opsin-stabilizing compound in a unit ofweight or volume suitable for administration to a subject. Thecompositions and combinations of the invention can be part of apharmaceutical pack, where each of the compounds is present inindividual dosage amounts.

The phrase “pharmaceutically acceptable” refers to those compounds ofthe present invention, compositions containing such compounds, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Non-oral pharmaceutical compositions of the invention to be used forprophylactic or therapeutic administration should be sterile. Sterilityis readily accomplished by filtration through sterile filtrationmembranes (e.g., 0.2 μm membranes), by gamma irradiation, or any othersuitable means known to those skilled in the art. Therapeuticopsin-binding or opsin-stabilizing compound compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. These compositions ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. The compounds may be combined, optionally, with apharmaceutically acceptable excipient.

The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction that wouldsubstantially impair the desired pharmaceutical efficacy.

Compounds of the present invention can be contained in apharmaceutically acceptable excipient. The excipient preferably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetate, lactate, tartrate, and otherorganic acids or their salts; tris-hydroxymethylaminomethane (TRIS),bicarbonate, carbonate, and other organic bases and their salts;antioxidants, such as ascorbic acid; low molecular weight (for example,less than about ten residues) polypeptides, e.g., polyarginine,polylysine, polyglutamate and polyaspartate; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), andpolyethylene glycols (PEGs); amino acids, such as glycine, glutamicacid, aspartic acid, histidine, lysine, or arginine; monosaccharides,disaccharides, and other carbohydrates including cellulose or, itsderivatives, glucose, mannose, sucrose, dextrins or sulfatedcarbohydrate derivatives, such as heparin, chondroitin sulfate ordextran sulfate; polyvalent metal ions, such as divalent metal ionsincluding calcium ions, magnesium ions and manganese ions; chelatingagents, such as ethylenediamine tetraacetic acid (EDTA); sugar alcohols,such as mannitol or sorbitol; counterions, such as sodium or ammonium;and/or nonionic surfactants, such as polysorbates or poloxamers. Otheradditives may be included, such as stabilizers, anti-microbials, inertgases, fluid and nutrient replenishers (i.e., Ringer's dextrose),electrolyte replenishers, which can be present in conventional amounts.

The compositions, as described above, can be administered in effectiveamounts. The effective amount will depend upon the mode oradministration, the particular condition being treated and the desiredoutcome. It may also depend upon the stage of the condition, the age andphysical condition of the subject, the nature of concurrent therapy, ifany, and like factors well known to the medical practitioner. Fortherapeutic applications, it is that amount sufficient to achieve amedically desirable result.

With respect to a subject suffering from, or at risk of developing,light toxicity, such as that due to ocular surgery, an effective amountis an amount sufficient to reduce the rate or extent of formation andaccumulation of visual cycle products, such as all-trans-retinal, orlipofuscin, or A2E as well as preventing photocell apoptosis as a resultof excessive rhodopsin activation. Here, the compounds of the presentinvention would be from about 0.01 mg/kg per day to about 1000 mg/kg perday. It is expected that doses ranging from about 50 to about 2000 mg/kgwill be suitable. Lower doses will result from certain forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of a composition of the present invention.

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. In one preferred embodiment, acomposition of the invention is administered intraocularly. Other modesof administration include oral, rectal, topical, intraocular, buccal,intravaginal, intracisternal, intracerebroventricular, intratracheal,nasal, transdermal, within/on implants, or parenteral routes.Compositions comprising a composition of the invention can be added to aphysiological fluid, such as to the intravitreal humor. For CNSadministration, a variety of techniques are available for promotingtransfer of the therapeutic across the blood brain barrier includingdisruption by surgery or injection, drugs which transiently openadhesion contact between the CNS vasculature endothelial cells, andcompounds that facilitate translocation through such cells. Oraladministration can be preferred for prophylactic treatment because ofthe convenience to the patient as well as the dosing schedule.

Pharmaceutical compositions of the invention can optionally furthercontain one or more additional proteins as desired, including plasmaproteins, proteases, and other biological material, so long as it doesnot cause adverse effects upon administration to a subject. Suitableproteins or biological material may be obtained from human or mammalianplasma by any of the purification methods known and available to thoseskilled in the art; from supernatants, extracts, or lysates ofrecombinant tissue culture, viruses, yeast, bacteria, or the like thatcontain a gene that expresses a human or mammalian plasma protein whichhas been introduced according to

standard recombinant DNA techniques; or from the fluids (e.g., blood,milk, lymph, urine or the like) or transgenic animals that contain agene that expresses a human plasma protein which has been introducedaccording to standard transgenic techniques.

Pharmaceutical compositions of the invention can comprise one or more pHbuffering compounds to maintain the pH of the formulation at apredetermined level that reflects physiological pH, such as in the rangeof about 5.0 to about 8.0 (e.g., 6.0, 6.5, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.8). The pH buffering compound used in the aqueousliquid formulation can be an amino acid or mixture of amino acids, suchas histidine or a mixture of amino acids such as histidine and glycine.Alternatively, the pH buffering compound is preferably an agent whichmaintains the pH of the formulation at a predetermined level, such as inthe range of about 5.0 to about 8.0, and which does not chelate calciumions. Illustrative examples of such pH buffering compounds include, butare not limited to, imidazole and acetate ions. The pH bufferingcompound may be present in any amount suitable to maintain the pH of theformulation at a predetermined level.

Pharmaceutical compositions of the invention can also contain one ormore osmotic modulating agents, i.e., a compound that modulates theosmotic properties (e.g., tonicity, osmolality and/or osmotic pressure)of the formulation to a level that is acceptable to the blood stream andblood cells of recipient individuals. The osmotic modulating agent canbe an agent that does not chelate calcium ions. The osmotic modulatingagent can be any compound known or available to those skilled in the artthat modulates the osmotic properties of the formulation. One skilled inthe art may empirically determine the suitability of a given osmoticmodulating agent for use in the inventive formulation. Illustrativeexamples of suitable types of osmotic modulating agents include, but arenot limited to: salts, such as sodium chloride and sodium acetate;sugars, such as sucrose, dextrose, and mannitol; amino acids, such asglycine; and mixtures of one or more of these agents and/or types ofagents. The osmotic modulating agent(s) maybe present in anyconcentration sufficient to modulate the osmotic properties of theformulation.

Compositions comprising an opsin-binding or opsin-stabilizing compoundof the present invention can contain multivalent metal ions, such ascalcium ions, magnesium ions and/or manganese ions. Any multivalentmetal ion that helps stabilize the composition and that will notadversely affect recipient individuals may be used. The skilled artisan,based on these two criteria, can determine suitable metal ionsempirically and suitable sources of such metal ions are known, andinclude inorganic and organic salts.

Pharmaceutical compositions of the invention can also be a non-aqueousliquid formulation. Any suitable non-aqueous liquid may be employed,provided that it provides stability to the active agents (a) containedtherein. Preferably, the non-aqueous liquid is a hydrophilic liquid.Illustrative examples of suitable non-aqueous liquids include: glycerol;dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols,such as ethylene glycol, diethylene glycol, triethylene glycol,polyethylene glycol (“PEG”) 200, PEG 300, and PEG 400; and propyleneglycols, such as dipropylene glycol, tripropylene glycol, polypropyleneglycol (“PPG”) 425, PPG 725, PPG 1000, PEG 2000, PEG 3000 and PEG 4000.

Pharmaceutical compositions of the invention can also be a mixedaqueous/non-aqueous liquid formulation. Any suitable non-aqueous liquidformulation, such as those described above, can be employed along withany aqueous liquid formulation, such as those described above, providedthat the mixed aqueous/non-aqueous liquid formulation provides stabilityto the compound contained therein. Preferably, the non-aqueous liquid insuch a formulation is a hydrophilic liquid. Illustrative examples ofsuitable non-aqueous liquids include: glycerol; DMSO; EMS; ethyleneglycols, such as PEG 200, PEG 300, and PEG 400; and propylene glycols,such as PPG 425, PPG 725, PEG 1000, PEG 2000, PEG 3000 and PEG 4000.Suitable stable formulations can permit storage of the active agents ina frozen or an unfrozen liquid state. Stable liquid formulations can bestored at a temperature of at least −70° C., but can also be stored athigher temperatures of at least 0° C., or between about 0° C. and about42° C., depending on the properties of the composition. It is generallyknown to the skilled artisan that proteins and polypeptides aresensitive to changes in pH, temperature, and a multiplicity of otherfactors that may affect therapeutic efficacy.

In certain embodiments a desirable route of administration can be bypulmonary aerosol. Techniques for preparing aerosol delivery systemscontaining polypeptides are well known to those of skill in the art.Generally, such systems should utilize components that will notsignificantly impair the biological properties of the antibodies, suchas the paratope binding capacity (see, for example, Sciarra and Cutie,“Aerosols,” in Remington's Pharmaceutical Sciences 18th edition, 1990,pp 1694-1712; incorporated by reference). Those of skill in the art canreadily modify the various parameters and conditions for producingpolypeptide aerosols without resorting to undue experimentation.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of compositions of the invention, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as polylactides (U.S. Pat. No.3,773,919; European Patent No. 58,481), poly(lactide-glycolide),copolyoxalates polycaprolactones, polyesteramides, polyorthoesters,poiyhydroxybutyric acids, such as poly-D-(−)-3-hydroxybutyric acid(European Patent No. 133,988), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, K R. et at, Biopolymers 22: 547-556),poly (2-hydroxyethyl methacrylate) or ethylene vinyl acetate (Langer, etal., J. Biomed. Mater. Res. 15:267-277; Langer, B. Chem. Tech.12:98-105), and polyanhydrides.

Other examples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Delivery systems also include non-polymer systems thatare: lipids including sterols such as cholesterol, cholesterol estersand fatty acids or neutral fats such as mono-, di- and tri-glycerides;hydrogel release systems such as biologically-derived bioresorbablehydrogel (i.e., chitin hydrogels or chitosan hydrogels); sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially filled implants; and thelike. Specific examples include, but are not limited to: (a) aerosionalsystems in which the agent is contained in a form within a matrix suchas those described in 13.5. U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034 and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253, and 3,854,480.

Another type of delivery system that can be used with the methods andcompositions of the invention is a colloidal dispersion system.Colloidal dispersion systems include lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vessels, which are useful as adelivery vector in vivo or in vitro. Large unilamellar vessels (LUV),which range in size from 0.2-4.0 μm, can encapsulate largemacromolecules within the aqueous interior and be delivered to cells ina biologically active form (Fraley, R., and Papahadjopoulos, D., TrendsBiochem. Sci. 6: 77-80).

Liposomes can be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Liposomes are commercially available from GibcoBRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed ofcationic lipids such as N-[1-(2, 3dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications, for example, in DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. (USA) 82:3688-3692 (1985); K. Hwang et al., Proc. Natl, Acad.Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Liposomes also have beenreviewed by Gregoriadis, G., Trends Biotechnol., 3: 235-241.

Another type of vehicle is a biocompatible microparticle or implant thatis suitable for implantation into the mammalian recipient. Exemplarybioerodible implants that are useful in accordance with this method aredescribed in PCT International application no. PCTIUS/03307 (PublicationNo—WO 95/24929, entitled “Polymeric Gene Delivery System”). PCT/US/0307describes biocompatible, preferably biodegradable polymeric matrices forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrices can be used to achieve sustainedrelease of the exogenous gene or gene product in the subject.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein an agent is dispersed throughout a solidpolymeric matrix) or a microcapsule (wherein an agent is stored in thecore of a polymeric shell). Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Other forms of the polymeric matrix for containing an agent includefilms, coatings, gels, implants, and stents. The size and composition ofthe polymeric matrix device is selected to result in favorable releasekinetics in the tissue into which the matrix is introduced. The size ofthe polymeric matrix further is selected according to the method ofdelivery that is to be used. Preferably, when an aerosol route is usedthe polymeric matrix and composition are encompassed in a surfactantvehicle. The polymeric matrix composition can be selected to have bothfavorable degradation rates and also to be formed of a material, whichis a bioadhesive, to further increase the effectiveness of transfer. Thematrix composition also can be selected not to degrade, but rather torelease by diffusion over an extended period of time. The deliverysystem can also be a biocompatible microsphere that is suitable forlocal, site-specific delivery. Such microspheres are disclosed inChickering, D. B., et al., Biotechnot. Bioeng, 52: 96-101; Mathiowitz,B., et at., Nature 386: 410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compositions of the invention to the subject. Suchpolymers may be natural or synthetic polymers. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

Exemplary synthetic polymers which can be used to form the biodegradabledelivery system include: polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluoses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulfate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate),poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene, poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate),poly(vinyl chloride), polystyrene, poly(vinylpyrrolidone), and polymersof lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone),and natural polymers such as alginate and other polysaccharidesincluding dextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof. In general, these materials degrade either byenzymatic hydrolysis or exposure to water in vivo, by surface or bulkerosion.

Methods of Ocular Delivery

The compositions of the invention are particularly suitable for treatingocular diseases or conditions, such as light toxicity, in particularlight toxicity related to an ocular surgical procedure.

In one approach, the compositions of the invention are administeredthrough an ocular device suitable for direct implantation into thevitreous of the eye. The compositions of the invention may be providedin sustained release compositions, such as those described in, forexample, U.S. Pat. Nos. 5,672,659 and 5,595,760. Such devices are foundto provide sustained controlled release of various compositions to treatthe eye without risk of detrimental local and systemic side effects. Anobject of the present ocular method of delivery is to maximize theamount of drug contained in an intraocular device or implant whileminimizing its size in order to prolong the duration of the implant.See, e.g., U.S. Pat. Nos. 5,378,475; 6,375,972, and 6,756,058 and U.S.Publications 20050096290 and 200501269448. Such implants may bebiodegradable and/or biocompatible implants, or may be non-biodegradableimplants.

Biodegradable ocular implants are described, for example, in U.S. PatentPublication No. 20050048099. The implants may be permeable orimpermeable to the active agent, and may be inserted into a chamber ofthe eye, such as the anterior or posterior chambers or may be implantedin the sclera, transchoroidal space, or an avascularized region exteriorto the vitreous. Alternatively, a contact lens that acts as a depot forcompositions of the invention may also be used for drug delivery.

In a preferred embodiment, the implant may be positioned over anavascular region, such as on the sclera, so as to allow for transcleraldiffusion of the drug to the desired site of treatment, e.g. theintraocular space and macula of the eye. Furthermore, the site oftranscleral diffusion is preferably in proximity to the macula. Examplesof implants for delivery of a composition of the invention include, butare not limited to, the devices described in U.S. Pat. Nos. 3,416,530;3,828,777; 4,014,335; 4,300,557; 4,327,725; 4,853,224; 4,946,450;4,997,652; 5,147,647; 164,188; 5,178,635; 5,300,114; 5,322,691;5,403,901; 5,443,505; 5,466,466; 5,476,511; 5,516,522; 5,632,984;5,679,666; 5,710,165; 5,725,493; 5,743,274; 5,766,242; 5,766,619;5,770,592; 5,773,019; 5,824,072; 5,824,073; 5,830,173; 5,836,935;5,869,079, 5,902,598; 5,904,144; 5,916,584; 6,001,386; 6,074,661;6,110,485; 6,126,687; 6,146.366; 6,251,090; and 6,299,895, and in WO01/30323 and WO 01/28474, all of which are incorporated herein byreference.

Examples include, but are not limited to the following: a sustainedrelease drug delivery system comprising an inner reservoir comprising aneffective amount of an agent effective in obtaining a desired local orsystemic physiological or pharmacological effect, an inner tubeimpermeable to the passage of the agent, the inner tube having first andsecond ends and covering at least a portion of the inner reservoir, theinner tube sized and formed of a material so that the inner tube iscapable of supporting its own weight, an impermeable member positionedat the inner tube first end, the impermeable member preventing passageof the agent out of the reservoir through the inner tube first end, anda permeable member positioned at the inner tube second end, thepermeable member allowing diffusion of the agent out of the reservoirthrough the inner tube second end; a method for administering a compoundof the invention to a segment of an eye, the method comprising the stepof implanting a sustained release device to deliver the compound of theinvention to the vitreous of the eye or an implantable, sustainedrelease device for administering a compound of the invention to asegment of an eye; a sustained release drug delivery device comprising:a) a drug core comprising a therapeutically effective amount of at leastone first agent effective in obtaining a diagnostic effect or effectivein obtaining a desired local or systemic physiological orpharmacological effect; b) at least one unitary cup essentiallyimpermeable to the passage of the agent that surrounds and defines aninternal compartment to accept the drug core, the unitary cup comprisingan open top end with at least one recessed groove around at least someportion of the open top end of the unitary cup; c) a permeable plugwhich is permeable to the passage of the agent, the permeable plug ispositioned at the open top end of the unitary cup wherein the grooveinteracts with the permeable plug holding it in position and closing theopen top end, the permeable plug allowing passage of the agent out ofthe drug core, though the permeable plug, and out the open top end ofthe unitary cup; and d) at least one second agent effective in obtaininga diagnostic effect or effective in obtaining a desired local orsystemic physiological or pharmacological effect; or a sustained releasedrug delivery device comprising: an inner core comprising an effectiveamount of an agent having a desired solubility and a polymer coatinglayer, the polymer layer being permeable to the agent, wherein thepolymer coating layer completely covers the inner core.

Other approaches for ocular delivery include the use of liposomes totarget a compound of the present invention to the eye, and preferably toretinal pigment epithelial cells and/or Bruch's membrane. For example,the compound maybe complexed with liposomes in the manner describedabove, and this compound/liposome complex injected into patients with anophthalmic condition, such as light toxicity, using intravenousinjection to direct the compound to the desired ocular tissue or cell.Directly injecting the liposome complex into the proximity of theretinal pigment epithelial cells or Bruch's membrane can also providefor targeting of the complex with some forms of ocular PCD. In aspecific embodiment, the compound is administered via intra-ocularsustained delivery (such as VITRASERT or ENVISION. In a specificembodiment, the compound is delivered by posterior subtenons injection.In another specific embodiment, microemulsion particles containing thecompositions of the invention are delivered to ocular tissue to take uplipid from Bruchs membrane, retinal pigment epithelial cells, or both.

Nanoparticles are a colloidal carrier system that has been shown toimprove the efficacy of the encapsulated drug by prolonging the serumhalf-life. Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymercolloidal drug delivery system that is in clinical development, asdescribed by Stella et al, J. Pharm. Sci., 2000. 89: p. 1452-1464;Brigger et al., Tnt. J. Pharm., 2001. 214: p. 37-42; Calvo et al.,Pharm. Res., 2001. 18: p. 1157-1166; and Li et al., Biol. Pharm. Bull.,2001. 24: p. 662-665. Biodegradable poly (hydroxyl acids), such as thecopolymers of poly (lactic acid) (PLA) and poly (lactic-co-glycolide)(PLGA) are being extensively used in biomedical applications and havereceived FDA approval for certain clinical applications. In addition,PEG-PLGA nanoparticles have many desirable carrier features including(i) that the agent to be encapsulated comprises a reasonably high weightfraction (loading) of the total carrier system; (ii) that the amount ofagent used in the first step of the encapsulation process isincorporated into the final carrier (entrapment efficiency) at areasonably high level; (iii) that the carrier have the ability to befreeze-dried and reconstituted in solution without aggregation; (iv)that the carrier be biodegradable; (v) that the carrier system be ofsmall size; and (vi) that the carrier enhance the particles persistence.

Nanoparticles are synthesized using virtually any biodegradable shellknown in the art. In one embodiment, a polymer, such as poly(lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used.Such polymers are biocompatible and biodegradable, and are subject tomodifications that desirably increase the photochemical efficacy andcirculation lifetime of the nanoparticle. In one embodiment, the polymeris modified with a terminal carboxylic acid group (COOH) that increasesthe negative charge of the particle and thus limits the interaction withnegatively charge nucleic acid aptamers. Nanoparticles are also modifiedwith polyethylene glycol (PEG), which also increases the half-life andstability of the particles in circulation. Alternatively, the COOH groupis converted to an N-hydroxysuccinimide (NHS) ester for covalentconjugation to amine-modified aptamers.

Biocompatible polymers useful in the composition and methods of theinvention include, but are not limited to, polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, poly(vinylpyrrolidone), polyglycolides,polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic esters, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, cellulosesulfate sodium salt poly-methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate\ poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate),polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate,polyvinyl chloride polystyrene, poly(vinyl pyrrolidone), polyhyaluronicacids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid,alginate, chitosan, poly(methyl methacrylates), poly(ethylmethacrylates), poly(butyl methacrylate), poly(isobutyl methacrylate),poly(hexyl methacrylate) poly(isodecyl methaerylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylatee), poly(isobutyl acrylate), poly(octadecylacrylate) and combinations of any of these, In one embodiment, thenanoparticles of the invention include PEG-PLGA polymers.

Compositions of the invention may also be delivered topically. Fortopical delivery, the compositions are provided in any pharmaceuticallyacceptable excipient that is approved for ocular delivery. Preferably,the composition is delivered in drop form to the surface of the eye. Forsome application, the delivery of the composition relies on thediffusion of the compounds through the cornea to the interior of theeye.

Those of skill in the art will recognize that treatment regimens forusing the compounds of the present invention to treat light toxicity orother opthalmic conditions (e.g., RP) can be straightforwardlydetermined. This is not a question of experimentation, but rather one ofoptimization, which is routinely conducted in the medical arts. In vivostudies in nude mice often provide a starting point from which to beginto optimize the dosage and delivery regimes. The frequency of injectionwill initially be once a week, as has been done in some mice studies.However, this frequency might be optimally adjusted from one day toevery two weeks to monthly, depending upon the results obtained frontthe initial clinical trials and the needs of a particular patient.

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. For certain embodiments it is envisioned that the dosage mayvary from between about 1 mg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose maybe about 1, 5, 10, 25, 50, 75,100, 150, 10 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, 5000 mg/Kg body weight. in other embodiments, it is envisaged thatlower does may be used, such doses may be in the range of about 5 mgcompound/Kg body to about 20 mg compound/Kg body. In other embodimentsthe doses may be about 8, 10, 12, 14, 16 15 or 18 mg/Kg body weight. Ofcourse, this dosage amount may be adjusted upward or downward, as isroutinely done in such treatment protocols, depending on the results ofthe initial clinical trials and the needs of a particular patient.

Screening Assays

Useful compounds of the invention are compounds of the formula (I) thatreversibly bind to a native or mutated opsin protein, such as in or nearthe 11-cis-retinal binding pocket. The non bleachable or slowlybleachable pigment rhodopsins formed from these small molecule opsinbindings will prevent light toxicity related to, for example, theaccumulation of visual cycle products as well as apoptotic photocelldeath resulting from excessive rhodopsin stimulation. Such binding willcommonly inhibit, if not prevent, binding of retinoids, especially11-cis-retinal, to the binding pocket and thereby reduce formation ofvisual cycle products, such as all-trans-retinal. Any number of methodsare available for carrying out screening assays to identify suchcompounds. In one approach, an opsin protein is contacted with acandidate compound or test compound that is a non-retinoid in thepresence of 11-cis-retinal or retinoid analog and the rate or yield offormation of chromophore is determined. If desired, the binding of thenon-retinoid to opsin is characterized. Preferably, the non-retinoidbinding to opsin is non-covalent and reversible. Thus, inhibition ofrhodopsin formation by a non-retinoid indicates identification of asuccessful test compound. An increase in the amount of rhodopsin isassayed, for example, by measuring the protein's absorption at acharacteristic wavelength (e.g., 498 nm for rhodopsin) or by measuringan increase in the biological activity of the protein using any standardmethod (e.g., enzymatic activity association with a ligand). Usefulcompounds inhibit binding of 11-cis-retinal (and formation of rhodopsin)by at least about 10%, 15%, or 20%, or preferably by 25%, 50%, or 75%,or most preferably by up to 90% or even 100%.

Alternatively, the efficacy of compounds useful in the methods of theinvention may be determined by exposure of a mammalian eye to a highintensity light source prior to, during, or following administration ofa test compound, followed by determination of the amount of visual cycleproducts (e.g., all-trans retinal, A2E, or lipofuscin) formed as aresult of exposure to the high intensity light source, wherein acompound of the invention will have reduced the amount of visual cycleproducts related to the exposure.

In sum, preferred test compounds identified by the screening methods ofthe invention are non-retinoids, are selective for opsin and bind in areversible, non-covalent manner to opsin protein. In addition, theiradministration to transgenic animals otherwise producing increasedlipofuscin results in a reduced rate of production or a reducedaccumulation of lipofuscin in the eye of said animal. Compoundsidentified according to the methods of the invention are useful for thetreatment of light toxicity or other ophthalmic condition in a subject,such as a human patient.

Compositions of the invention useful for the prevention of lighttoxicity, as well as AMD and retinitis pigmentosa, can optionally becombined with additional therapies as heretofore described.

Examples

The following non-limiting examples further describe and enable one ofordinary skill in the art to make use of the invention.

Example 1:(±)-(3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

A mixture of (E)-1-(2,6,6-trimethylcyclohex-1-enyl)but-2-en-1-one (400mg, 2.06 mmol) in phosphoric acid (85%, 3.0 mL) was stirred at roomtemperature for 3 hours. The mixture was poured into water (30 mL) andthe organics extracted with ethyl acetate (40 mL×2). The organic layerwas washed with brine, dried over anhydrous sodium sulfate and thenconcentrated under reduced pressure. The residue was purified by silicagel chromatography to give the title compound as a light yellow oil (362mg, Yield: 91%). R_(f)=0.6 (20:1 petroleum ether/ethyl acetate); ¹H NMR(400 MHz, CDCl₃) δ 5.75 (s, 1H), 1.98 (s, 3H), 1.82 (s, 1H), 1.64-1.52(m, 4H), 1.36-1.33 (m, 2H), 1.18 (s, 3H), 1.16 (s, 3H), 0.88 (s, 3H)ppm; Mass spectrum (ESI+ve) m/z 193 (M+H⁺).

Example 2: (±)-(3aS,7aR)-4,4-dimethylhexahydro-1H-indol-2(3H)-oneExample 2a: 1-benzyl-4,4-dimethyl-3a,4,5,6-tetrahydro-1H-indol-2(3H)-one

To a solution of ethyl 2-(2,2-dimethyl-6-oxocyclohexyl)acetate (300 mg,1.413 mmol) in 1,2-dichloroethane (2.5 mL) was added benzylamine (182mg, 1.7 mmol), acetic acid (170 mg, 2.83 mmol) and sodiumtriacetoxyborohydride (381 mg, 1.8 mmol). The mixture was stirred atroom temperature for 48 hours. The reaction mixture was diluted withdichloromethane (10 mL) and saturated aqueous sodium bicarbonate (5 mL)and the layers were separated. The aqueous layer was extracted withdichloromethane (3×10 mL) and the combined organic phase was dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to afford thetitle compound as a light yellow oil (88 mg, Yield: 19%). ¹H NMR (400MHz, CDCl₃) δ 7.34-7.21 (m, 5H), 4.78 (d, J=15.2 Hz, 1H), 4.75-4.72 (m,1H), 4.51 (d, J=16.0 Hz, 1H), 2.78-2.61 (m, 1H), 2.52-2.24 (m, 3H),1.56-1.40 (m, 3H), 1.01 (s, 3H), 0.85 (s, 3H) ppm; Mass spectrum(ESI+ve) m/z 256 (M+H⁺).

Example 2b:(±)-(3aS,7aR)-1-benzyl-4,4-dimethylhexahydro-1H-indol-2(3H)-one

To a solution of the product of Example 2a (88 mg, 0.35 mmol) inmethanol (3 mL) was added wet 10% Pd/C (10 mg). The mixture was stirredunder an atmosphere of hydrogen overnight and then was suction filteredand the filtrate was concentrated under reduced pressure. The residuewas purified by silica gel column chromatography to give the titlecompound as a colorless oil (46 mg, Yield: 52%). ¹H NMR (400 MHz, CDCl₃)δ 7.37-7.29 (m, 3H), 7.25 (d, J=7.2 Hz, 2H), 4.94 (d, J=15.6 Hz, 1H),3.96 (d, J=14.8 Hz, 1H), 3.44-3.32 (m, 1H), 2.38-2.20 (m, 2H), 2.06-1.90(m, 2H), 1.51-1.48 (m, 1H), 1.32-1.22 (m, 3H), 1.10-0.97 (m, 1H), 0.93(s, 3H), 0.91 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 258 (M+H⁺).

Example 2: (±)-(3aS,7aR)-4,4-dimethylhexahydro-1H-indol-2(3/1)-one

To a solution of the product of Example 2b (25 mg, 0.098 mmol) in liquidammonia (20 mL) was added lithium (50 mg). The mixture was stirred at−50° C. for 2 hours. Ethanol (10 mL) was slowly added to the reactionmixture at −50° C. and then it was allowed to warm slowly to roomtemperature. The reaction mixture was concentrated under reducedpressure and the residual solid was dissolved in water (10 mL) and thenit was extracted with ethyl acetate (3×20 mL). The combined organicphase was washed with brine (5 mL), dried over anhydrous magnesiumsulfate, filtered and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography to afford the titlecompound a colorless oil (13 mg, Yield: 79%). ¹H NMR (400 MHz, CDCl₃) δ5.77 (brs, 1H), 3.60-3.49 (m, 1H), 2.26-2.04 (m, 3H), 1.94-1.82 (m, 1H),1.54-1.49 (m, 1H), 1.45-1.17 (m, 5H), 0.99 (s, 3H), 0.92 (s, 3H) ppm;Mass spectrum (ESI+ve) m/z 168 (M+H⁺).

Example 3:(±)-(3aS,7aS)-4,4,7,7-tetramethylhexahydrobenzofuran-2(3H)-one Example3a: 1,4,4-trimethylcyclohex-2-enol

To a stirred solution of 4,4-dimethylcyclohex-2-enone (40.0 g, 322 mmol)in 400 ml of anhydrous ether at −78° C. was added drop wise an etherealsolution of methyl lithium (220 ml of a 1.6 M ethereal solution). Theresulting solution was allowed to warm to room temperature and stirredfor 18 hours. The reaction was quenched by the addition of water (200mL). The phases were separated and the aqueous layer extracted withdiethyl ether (2×200 mL). The combined organic phase was washed withbrine (2×50 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure to give the title compound as a light yellow oil(41 g, Yield: 90%). R_(f)=0.5 (5:1 petroleum ether/ethyl acetate); ¹HNMR (400 MHz, CDCl₃) δ 5.46 (d, J=10.0 Hz, 1H), 5.43 (d, J=10.0 Hz, 1H),1.73-1.70 (m, 2H), 1.59-1.56 (m, 1H), 1.50-1.45 (m, 1H), 1.27 (s, 3H),1.01 (s, 3H), 0.95 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 123(M−H₂O+H⁺).

Example 3b: 3,6,6-trimethylcyclohex-2-enone

To a stirred slurry of pyridinium chlorochromate (123 g, 570 mmol), indichloromethane (840 mL) at room temperature was added in one portion asolution of the product of Example 3a (40.0 g, 285 mmol) indichloromethane (240 mL). The resulting dark red mixture was allowed tostir for 18 hours at room temperature after which it was filtered andthe precipitate washed with diethyl ether (200 mL). The filtrate waswashed successively with 5% aqueous sodium hydroxide (2×200 mL), 5%aqueous hydrochloric acid (200 mL) and saturated aqueous sodiumbicarbonate (2×50 mL). The organic phase was dried over anhydrousmagnesium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to afford thetitle compound as a colorless oil (14 g, Yield: 35%). R_(f)=0.4 (5:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 5.77 (s, 1H),2.29 (t, J=6.0 Hz, 2H), 1.93 (s, 3H), 1.80 (d, J=6.0 Hz, 3H), 1.09 (s,6H) ppm; Mass spectrum (ESI+ve) m/z 139 (M+H⁺).

Example 3c: 2,2,5,5-tetramethylcyclohexanone

Cuprous iodide (6.9 g, 36.2 mmol) was added to a dry 250-mL round-bottomflask equipped with a stir bar and sealed under argon with a septum. Theflask was evacuated with a vacuum pump and purged with argon. Thisprocess was repeated three times. Tetrahydrofuran (75 mL) was injectedand the slurry was cooled to −78° C. and then methyl lithium (45 mL, 72mmol) was added drop wise. The mixture was allowed to warm untilhomogeneous and then was recooled to −78° C. and boron trifluorideetherate (8.9 mL, 72 mmol) was added via a syringe. The compound ofExample 3b (5.0 g, 36.2 mmol) was added neat and the reaction mixturewas stirred for 1.5 hours. The reaction was quenched with ammoniumhydroxide/saturated ammonium chloride (1:9, 250 mL). The organics wereextracted with ethyl acetate (250 mL) and the organic layer was washedwith aqueous saturated sodium bicarbonate (50 mL×2), brine (50 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto afford the title compound as a clear oil (1.5 g, Yield: 26%). ¹H NMR(400 MHz, CDCl₃) δ 2.21 (s, 2H), 1.69-1.65 (m, 2H), 1.61-1.57 (m, 2H),1.09 (s, 6H), 0.94 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 216.36, 51.32,44.00, 36.89, 36.62, 34.69, 28.5, 25.15 ppm; Mass spectrum (ESI+ve) m/z155 (M+H⁺).

Example 3d: ethyl 2-(2,2,5,5-tetramethyl-6-oxocyclohexyl)acetate

To a solution of the product of Example 3c (308 mg, 2.0 mmol) intetrahydrofuran (5 mL) at −78° C. was added lithium diisopropylamide(1.1 mL, 2.2 mmol) and the solution was stirred for 1 hour. Ethyl2-bromoacetate (680 mg, 4.0 mmol) and hexamethylphosphoramide (427 mg,2.4 mmol) was added and the solution was allowed to warm to roomtemperature and stirred overnight. The mixture was poured into saturatedammonium chloride and the organics were extracted with ethyl acetate(150 mL). The organic layer was washed with water (100 ml×2) and brine(50 mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound as a colorless oil (200 mg)that was used directly in the next step.

Example 3:(±)-(3aS,7aS)-4,4,7,7-tetramethylhexahydrobenzofuran-2(3H)-one

To a mixture of the product of Example 3d (180 mg, 0.4 mmol) in methanol(2.0 mL) at 0° C. was added sodium borohydride (38 mg, 1.0 mmol). Thereaction mixture was stirred for 2 hours and then the reaction wasquenched with saturated aqueous ammonium chloride. The organics wereextracted with ethyl acetate (150 mL) and the organic layer was washedwith water (100 mL×2), brine (50 mL×2). dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound as a white solid (10 mg, Yield: 13%). Mp=94.8-95.2° C.;R_(f)=0.4 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃)δ 4.03 (d, J=5.2 Hz, 1H), 2.44 (d, J=5.2 Hz, 2H), 2.22-2.19 (m, 1H),1.39-1.26 (m, 4H), 1.07 (s, 3H), 1.01 (s, 3H), 0.94 (s, 3H), 0.91 (s,3H) ppm; Mass spectrum (ESI+ve) m/z 197 (M+H⁺).

Example 4: (±)-(3aS,7aR)-1,4,4-trimethylhexahydro-1H-indol-2(3H)-oneExample 4a: 1,4,4-trimethyl-3a,4,5,6-tetrahydro-1H-indol-2(3H)-one

To a solution of ethyl 2-(2,2-dimethyl-6-oxocyclohexyl)acetate (300 mg,1.4 mmol) in 1,2-dichloroethane (3 mL) was added methylamine alcoholsolution (340 mg, 2.83 mmol), sodium triacetoxyborohydride (390 mg, 1.84mmol) and acetic acid (0.17 mL, 2.83 mmol). The mixture was stirred atroom temperature overnight. Water (10 mL) was added to the reactionmixture and then the organics were extracted with ethyl acetate (3×30mL). The organic layer was washed with brine (5 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to give thetitle compound as a light yellow oil (40 mg, Yield: 16%). ¹H NMR (400MHz, CDCl₃) δ 4.72 (dd, J=6.4, 3.2 Hz, 1H), 2.86 (s, 3H), 2.63-2.53 (m,1H), 2.33 (dd, J=16.4, 9.2 Hz, 1H), 2.22-2.08 (m, 3H), 1.54-1.41 (m,2H), 0.94 (s, 3H), 0.78 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 180(M+H⁺).

Example 4: (±)-(3aS,7aR)-1,4,4-trimethylhexahydro-1H-indol-2(3H)-one

To a solution of the product of Example 4a (40 mg, 0.223 mmol) inmethanol (4 mL) was added Pd/C (10 mg). The mixture was stirred under ahydrogen atmosphere overnight. The reaction mixture was suction filteredand the filtrate was concentrated under reduced pressure. The residuewas purified by silica gel column chromatography to afford the titlecompound as a colorless oil (36 mg, Yield: 89%). R_(f)=0.2 (3:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 3.49-3.36 (m,1H), 2.78 (s, 3H), 2.25-2.10 (m, 2H), 2.10-1.98 (m, 2H), 1.54-1.49 (m,1H), 1.37-1.21 (m, 3H), 1.03-0.96 (m, 1H), 0.92 (s, 3H), 0.86 (s, 3H)ppm; Mass spectrum (ESI+ve) m/z 182 (M+H⁺).

Example 5: (±)-(3aS,7aR)-4,4,7a-trimethyloctahydro-1H-inden-1-oneExample 5a: 2-methyl-2-(3-oxopentyl)cyclopentane-1,3-dione

To a solution of 2-methylcydopentane-1,3-dione (20 g, 178.2 mmol) andpent-1-en-3-one (15 g, 178.2 mmol) in dimethoxyethane (400 mL) was added1,4-diazabicyclo[2.2.2]octane (20 g, 178.2 mmol). The mixture wasstirred for 24 hours at room temperature. The mixture was acidified with1N hydrochloric acid to pH=4.5 and the organics were extracted withdiethyl ether (200 mL×3). The organic layer was washed with brine (100mL×3) dried over anhydrous sodium sulfate and concentrated under reducedpressure to give the title compound as a light yellow oil (21.9 g,Yield: 63%). ¹H NMR (400 MHz, CDCl₃) δ 2.91-2.69 (m, 4H), 2.46-2.34 (m,4H), 1.90 (t, J=7.2 Hz, 2H), 1.11 (s, 3H), 1.01 (t, J=7.4 Hz, 3H) ppm;Mass spectrum (ESI+ve) m/z 197 (M+H⁺).

Example 5b: 4,7a-dimethyl-2,3,7,7a-tetrahydro-1H-indene-1,5(6H)-dione

To a solution of the product of Example 5a (21.92 g, 111.7 mmol) intoluene (250 mL) was added p-toluenesulfonic acid (6.37 g, 33.51 mmol).The mixture was refluxed for 1 hour. The mixture was concentrated underreduced pressure and the residue was extracted with dichloromethane (100mL×3). The organic layer was washed with saturated aqueous sodiumbicarbonate (100 mL×2) and brine (100 mL×2), dried over anhydrous sodiumsulfate and concentrated under reduced pressure to give the titlecompound as a brown yellow oil (20 g, yield: 100%). ¹H NMR (400 MHz,CDCl₃) δ 3.00-2.69 (m, 3H), 2.60-2.36 (m, 3H), 2.07 (ddd, J=13.4, 5.2,2.2 Hz, 1H), 1.90-1.79 (m, 1H), 1.77 (s, 3H), 1.28 (s, 3H) ppm; Massspectrum (ESI+ve) m/z 179 (M+H⁺).

Example 5c:4′,7a′-dimethyl-2′,3′,7′,7a′-tetrahydrospiro[[1,3]dioxolane-2,1′-inden]-5′6′H)-one

To a solution of the product of Example 5b (5 g, 28.0 mmol) in2-ethyl-2-methyl-1,3-dioxolane (21 mL) was added p-toluenesulfonic acid(373 mg, 1.96 mmol). The mixture was stirred at room temperature for 3days. The reaction was quenched by the addition of a few drops oftriethylamine. Benzene (50 mL) was added and the solution was washedwith water (20 mL×2). The organic layer was washed with saturatedaqueous sodium bicarbonate (20 mL×2) and brine (20 mL×2), dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel flash chromatography to give thetitle compound as yellow oil (4.02 g, Yield: 64%). ¹H NMR (400 MHz,DMSO) δ 4.05-3.90 (m, 4H), 2.62-2.41 (m, 4H), 2.33-2.15 (m, 2H),1.99-1.90 (m, 1H), 1.69 (s, 3H), 1.61 (ddd, J=12.6, 5.4, 2.0 Hz, 1H),1.27 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 223 (M+H⁺).

Example 5d: (±)-(3a'S,7a′R)-4′,4′,7a′-trimethylhexahydrospiro[[1,3]dioxolane-2,1′-inden]-5′(6′H)-one

A solution of the product of Example 5c (3.0 g, 13.5 mmol) in anhydroustetrahydrofuran (33 mL) was added drop wise to a solution of lithium(300 mg, 43.2 mmol) in ammonia (189 mL) at −78° C. After the solutionwas stirred for 1 hour, methyl iodide (4.5 mL, 72.9 mmol) was added dropwise. After 3 hours at −78° C., the reaction was allowed to warm to roomtemperature and stirring was continued overnight. Water (45 mL) wasadded and the mixture was extracted with diethyl ether (60 mL×2). Theorganic layer was washed with water (50 mL×2) and brine (50 mL×2), driedover anhydrous sodium sulfate and concentrated under reduced pressure.The residue was purified by silica gel flash chromatography to give thetitle compound as a light yellow solid (1.70 g, Yield: 53%). ¹H NMR (400MHz, CDCl₃) δ 4.06-3.81 (m, 4H), 2.80-2.64 (m, 1H), 2.33-2.20 (m, 1H),2.09-2.00 (m, 1H), 1.97-1.68 (m, 5H), 1.32-1.26 (m, 1H), 1.29 (s, 3H),1.26 (s, 3H), 1.01 (s, 3H); ¹³C NMR (100 MHz, DMSO) δ 217.26, 120.03,65.50, 64.26, 55.87, 47.33, 45.10, 34.79, 31.97, 30.74, 27.42, 24.43,23.32, 21.27 ppm; Mass spectrum (ESI+ve) m/z 239 (M+H⁺).

Example 5e:(±)-(3a'S,7a′R)-4′,4′,7a′-trimethyloctahydrospiro[[1,3]dioxolane-2,1′-indene]

A mixture of the product of Example 5d (500 mg, 2.1 mmol), potassiumhydroxide (943 mg, 16.8 mmol) and hydrazine hydrate (1.03 mL, 21 mmol)in diethylene glycol (12 mL) was heated at 210° C. for 2 hours. Excesshydrazine was removed and the reaction mixture was heated for anadditional 12 hours. After dilution with water (40 mL), the mixtureextracted with diethyl ether (40 mL×2). The organic layer was washedwith brine (30 mL×2), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by flashchromatography to give 260 mg of a colorless oil which was furtherpurified by prep-TLC to give the title compound as a colorless oil (138mg, Yield: 29%). ¹H NMR (400 MHz, DMSO) δ 3.87-3.77 (m, 4H), 1.80-1.06(m, 11H), 0.99 (s, 3H), 0.92 (s, 3H), 0.78 (s, 3H); ¹³C NMR (100 MHz,DMSO) δ 120.57, 65.26, 63.61, 51.58, 45.39, 33.65, 31.72, 31.39, 30.73,29.21, 28.77, 21.96, 19.89, 18.60 ppm; Mass spectrum (ESI+ve) m/z 225(M+H⁺).

Example 5: (±)-(3aS,7aR)-4,4,7a-trimethylocthydro-1H-inden-1-one

To a solution of compound the product of Example 5e (60 mg, 0.27 mmol)in acetone (5 mL) was added p-toluenesulfonic acid (4.2 mg, 0.022 mmol).Then the mixture was refluxed for 1.5 hours. The mixture wasconcentrated and extracted with ethyl acetate (20 mL). The organic layerwas washed by saturated aqueous sodium bicarbonate (20 mL×2) and brine(20 mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by prep-TLC to give the titlecompound as a colorless oil (32 mg, Yield: 65%). R_(f)=0.7 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.47-2.38 (m,1H), 2.20-2.08 (m, 1H), 1.96-1.87 (m, 1H), 1.78-1.68 (m, 1H), 1.66-1.53(m, 2H), 1.50-1.25 (m, 4H), 1.21 (s, 3H), 1.17-1.10 (m, 1H), 1.08 (s,3H), 0.90 (s, 3H); ¹³C NMR (100 MHz, DMSO) δ 223.13, 53.28, 48.44,35.00, 35.02, 31.52, 29.52, 29.21, 28.70, 22.75, 21.37, 18.22 ppm; Massspectrum (ESI+ve) m/z 181 (M+H⁺).

Example 6: 4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-oneExample 6a: ethyl 2-(1,3,3-trimethyl-2-oxocyclohexyl)acetate

To a solution of 2,2,6-trimethylcyclohexanone (2.0 g, 14.26 mmol) in drytetrahydrofuran (15.0 mL) was added lithium diisopropylamide (7.8 mL,15.68 mmol) drop wise over 10 min at −78° C. and the resulting solutionwas allowed to warm to room temperature over 2 hours. The resultingmixture was recooled to −78° C. and hexamethylphosphoramide (2.7 g, 15.0mmol) was added. Then, ethyl bromoacetate (4.8 g, 28.52 mmol) intetrahydrofuran (6.0 mL) was added drop wise at −78° C. The resultingmixture was allowed to warm to room temperature and stirred for 4 hours.Saturated aqueous ammonium chloride (60 mL) was added and the mixturewas extracted with ethyl acetate (80 mL×2). The combined organic phasewas washed with brine (100 mL) and concentrated under reduced pressure.The residue was purified by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=100/1->70/1) to give the title compound asa colorless oil (3.0 g, Yield: 91%). ¹H NMR (400 MHz, CDCl₃) δ:4.12-4.04 (m, 2H), 3.03 (d, J=16.4 Hz, 1H), 2.14 (d, J=16.4 Hz, 1H),2.04 (s, 1H), 1.85-1.81 (m, 2H), 1.69-1.59 (m, 2H), 1.32-1.21 (m, 4H),1.14 (s, 9H) ppm; Mass spectrum (ESI+ve) m/z 227 (M+H⁺).

Example 6b: dimethyl 2-oxo-3-(1,3,3-trimethyl-2-oxocyclohexyl)propylphosphonate

To a solution of dimethyl methylphosphonate (163 mg, 1.32 mmol) in drytetrahydrofuran (4 mL) was added n-butyl lithium (0.82 mL, 1.32 mmol)drop wise at −60° C. and the solution was stirred for 30 minutes. Theproduct of Example 6a (100 mg, 0.44 mmol) in dry tetrahydrofuran (2.0mL) was added drop wise. The resulting mixture was allowed to warmslowly 0° C. and stirred for 4 hours. Acetic acid was added to thesolution to adjust to pH=7. Water (10 mL) was added to the solution andthe organics were extracted with ethyl acetate (20 mL×2). The combinedorganic phase was washed with brine (30 mL), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (eluent: petroleumether/ethyl acetate=1/3) to give the title compound as a colorless oil(20 mg, Yield: 15%). ¹H NMR (400 MHz, CDCl₃) δ 3.77 (d, J=6.0 Hz, 2H),3.75 (d, J=8.8 Hz, 1H), 3.30 (d, J=18.4 Hz, 1H), 3.10 (dd, J=22.4, 13.6Hz, 1H), 2.92 (dd, J=22:8, 13.6 Hz, 1H), 2.60 (d, J=18.4 Hz, 1H),1.92-1.76 (m, 3H), 1.67-1.51 (m, 3H), 1.15 (s, 3H), 1.12 (s, 6H) ppm;Mass spectrum (ESI+ve) m/z 305 (M+H⁺).

Example 6: 4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of the product of Example 6b (124 mg, 0.407 mmol) in drytetrahydrofuran (8.0 mL) was added sodium hydride (163 mg, 4.07 mmol) inone portion and stirred for 10 minutes at room temperature and then thereaction was heated to 60° C. for 3 hours. The reaction was quenchedwith the addition of saturated aqueous ammonium chloride (20 mL) andwater (10 mL). The organic were extracted with ethyl acetate (50 mL×2)and the combined organic extracts were washed with brine (50 mL), driedover anhydrous sodium sulfate and concentrated in vacuo. The residue waspurified by column silica gel column chromatography (eluent: petroleumether/ethyl acetate=5/1) to afford the title compound as a colorless oil(48 mg, Yield: 70%). R_(f)=0.6 (5:1 petroleum ether/ethyl acetate); ¹HNMR (400 MHz, CDCl₃) δ: 5.82 (s, 1H), 2.30 (s, 2H), 1.94-1.80 (m, 2H),1.69-1.57 (m, 2H), 1.43-1.32 (m, 5H), 1.25 (s, 3H), 1.20 (s, 3H) ppm;Mass spectrum (ESI+ve) m/z 179 (M+H⁺).

Example 7: 4,4-dimethylhexahydrobenzofuran-2(3H)-one

To a solution of ethyl 2-(2,2-dimethyl-6-oxocyclohexyl)acetate (200 mg,0.942 mmol) in methanol (5 mL) at 0° C. was added sodium borohydride(143 mg, 3.77 mmol) portionwise. The mixture was warmed to roomtemperature and stirred overnight. The reaction mixture was concentratedunder reduced pressure and the residue was partitioned between water andethyl acetate (2×20 mL). The combined organic phase was washed withbrine (5 mL), dried over anhydrous magnesium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to afford the title compound as a colorlessoil (100 mg, Yield: 63%). R_(f)=0.2 (20:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 4.64-4.56 (m, 1H), 2.49-2.19 (m,3H), 2.07-2.02 (m, 1H), 1.63-1.58 (m, 1H), 1.48-1.25 (m, 5H), 0.99 (s,3H), 0.93 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 169 (M+H⁺).

Example 8: (±)-(3aS,7aS)-methyl3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxylate Example8a: (±)-(3aS,7aR)-methyl4,4,7a-trimethyl-1-oxooctahydro-1H-indene-2-carboxylate

To a solution of lithium diisopropylamide (1.66 mL, 3.32 mmol) intetrahydrofuran (6 mL) at 0° C. under argon was drop wise added asolution of the product of Example 5 (300 mg, 1.66 mmol) intetrahydrofuran (6 mL). Then the solution was warmed to room temperatureand stirred for 45 minutes. The reaction mixture was cooled to 0° C. anddimethyl carbonate (1.40 mL, 16.6 mmol) was added. The mixture was thenwarmed room temperature and stirred for 6 hours. The reaction wasquenched with water and the organics extracted with ethyl acetate (100mL). The organic layer was washed with saturated ammonium chloride (50mL×2), water (50 mL×2) and brine (50 mL×2), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by prep-TLC to give the title compound as a yellow oil, whichwas confirmed as a mixture of stereo isomers (178 mg, Yield: 45%).R_(f)=0.3 (50:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃)(Major isomer) δ 3.78 (s, 3H), 3.19 (t, J=6.4 Hz, 1H), 2.18 (t, J=6.4Hz, 2H), 1.55-1.41 (m, 7H), 1.28 (s, 3H), 1.11 (s, 3H), 0.96 (s, 3H)ppm; Mass spectrum (ESI+ve) m/z 239 (M+H⁺).

Examples 8b and 8c: (±)-(1R,2R,3aR,7aS)-methyl1-hydroxy-4,4,7a-trimethyloctahydro-1H-indene-2-carboxylate (8b) and(±)-(1 S,2R,3aS,7aR)-methyl1-hydroxy-4,4,7a-trimethyloctahydro-1H-indene-2-carboxylate (8c)

To an ice-cooled solution of the product of Example 8a (100 mg, 0.42mmol) in tetrahydrofuran (5.2 mL) and methanol (0.6 mL) at 0° C. wasadded sodium borohydride (16 mg, 0.42 mmol) and the reaction mixture wasstirred for 4 hours. The reaction was quenched with 1N hydrochloric acidand extracted with diethyl ether (50 mL). The organic layer was washedwith brine (30 mL×2), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified byprep-TLC to give the title compound (8b) as a light yellow oil (32 mg,Yield: 32%). R_(f)=0.5 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 3.83-3.82 (m, 1H), 3.70 (s, 3H), 3.06-2.98 (m, 1H), 2.97(bs, 1H),1.97-1.87 (m, 2H), 1.53-1.50 (m, 2H), 1.43-1.36 (m, 2H),1.30-1.24 (m, 2H), 1.18-1.13 (m, 1H), 1.15 (s, 3H), 1.03 (s, 3H), 0.81(s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 175.9, 82.6, 51.8, 51.5, 43.6, 43.5,34.0, 31.8, 30.9, 29.7, 28.5, 25.5, 25.0, 18.5 ppm; Mass spectrum(ESI+ve) m/z 241 (M+H⁺). The title compound 8c light yellow oil (28 mg,Yield: 28%). R_(f)=0.4 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 3.78 (d, J=8.8 Hz, 1H), 3.71 (s, 3H), 2.74-2.67 (m, 1H),2.12 (bs, 1H), 1.89-1.81 (m, 2H), 1.53-1.40 (m, 3H); 1.31-1.18 (m, 3H),1.14 (s, 3H), 1.09-1.03 (m, 1H), 1.02 (s, 3H), 0.79 (s, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 176.8, 84.4, 52.0, 50.8, 46.9, 43.6, 33.9, 31.8,30.3, 28.3, 26.8, 26.1, 24.4, 18.2 ppm; Mass spectrum (ESI+ve) m/z 241(M+H⁺).

Example 8: (±)-(3aS,7aS)-methyl3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxylate

To a solution of the product of Example 8b (50 mg, 0.21 mmol) indichloromethane. (2 mL) was drop wise added thionyl chloride (64 mL) andpyridine (50 mL) and the reaction mixture was refluxed for 4 hours.Dichloromethane (30 mL) was added and the organic phase was washed withwater (30 ml×2) and brine (30 ml×2), dried over anhydrous sodium sulfateand concentrated under reduced pressure. Purification of the residue bysilica column column chromatography gave 44 mg of a colorless oil whichwas further purified by prep-TLC to afford the title compound as acolorless oil (23 mg, Yield: 48%). R_(f)=0.6 (100:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 6.56 (t, J=1.2 Hz, 1H),3.72 (s, 3H), 2.49-2.47 (m, 1H), 2.44-2.40 (m, 1H), 1.70-1:68 (m, 1H),1.51-1.27 (m, 5H), 1.24 (s, 3H), 1.14-1.10 (m, 1H), 1.02 (s, 3H), 0.91(s, 3H) ppm; Mass spectrum (ESI+ve) m/z 223 (M+H⁺).

Example 9: (±)-(3R,3aS,7aS)-3,3a,7,7-tetramethyloctahydro-1H-inden-1-one

To a solution of the product of Example 1 (400 mg, 2.08 mmol) inmethanol (5 mL) was added 10% Pd/C (80 mg) and the mixture was stirredunder an atmosphere of hydrogen for 24 hours. The reaction was suctionfiltered and the filtrate concentrated under reduced pressure to affordthe title compound as a colorless oil (364 mg, Yield: 91%). R_(f)=0.9(20:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.32(dd, J=18.4, 8.0 Hz, 1H), 1.91 (dd, J=18.0, 12.0 Hz, 1H), 1.85-1.77 (m,1H), 1.65-1.46 (m, 3H), 1.30-1.06 (m, 9H), 1.04 (s, 3H), 1.02-0.99 (m,1H), 0.96 (d, J=6.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 218.0, 65.7,44.2, 42.3, 38.9, 36.3, 33.1, 29.8, 29.5, 27.7, 25.3, 18.3, 12.3 ppm;Mass spectrum (ESI+ve) m/z 217 (M+Na⁺).

Example 10: 4,4,7a-trimethyl-2,4,5,6,7,7a-hexahydro-1H-inden-2-ol

To a solution of the product of Example 6 (120 mg, 0.67 mmol) in drytetrahydrofuran (12.0 mL) at 0° C. was added lithium aluminum hydride(77 mg, 2 mmol) in one portion. The resulting mixture was stirred at 0°C. for 1 hour. Water (10 mL) was added to the solution and the organicsextracted with ethyl acetate (50 mL×2). The organic phase was washedwith brine (50 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=8/1) to afford thetitle compound as a colorless oil (46 mg, Yield: 38%). R_(f)=0.4 (5:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 5.34 (s, 1H),4.89 (t, J=7.2 Hz, 1H), 2.24 (dd, J=12.0, 6.4 Hz, 1H), 1.78-1.65 (m,2H), 1.50-1.39 (m, 4H), 1.33-1.11 (m, 2H), 1.17 (s, 3H), 1.03 (s, 3H),1.01 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 163 (M−H₂O+H⁺).

Example 11: 2-methoxy-4,4,7a-trimethyl-2,4,5,6,7,7a-hexahydro-1H-indene

To a stirred solution of the product of Example 10 (46 mg, 0.25 mmol) indichloromethane (6.0 mL) at 0° C. was added1,8-bis(dimethylamino)naphthalene (535 mg, 2.5 mmol), followed bytrimethyloxonium tetrafluoroborate (370 mg, 2.5 mmol). The resultingmixture was warmed to room temperature and stirred for 2 hours. Thereaction was quenched by the addition of saturated aqueous sodiumbicarbonate (15 mL) and the organic phase was separated. The aqueousphase was extracted with dichloromethane (60 mL×2). The combined organicphase was washed with citric acid (60 mL), dried over anhydrous sodiumsulfate and concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: petroleum ether/ethyl acetate=80/1)to afford the title compound as a colorless oil (33 mg, Yield: 69%).R_(f)=0.6 (50:1 hexanes/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 5.44(d, J=0.8 Hz, 1H), 4.47 (dt, J=0.8, 7.6 Hz, 1H), 3.33 (s, 3H), 2.16 (dd,J=12.0, 6.4 Hz, 1H), 1.78-1.67 (m, 2H), 1.50-1.33 (m, 3H), 1.32-1.17 (m,2H), 1.17 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H) ppm; Mass spectrum(APCI+ve) m/z 163 (M−CH₃OH+H⁺).

Example 12: 7,7-dimethyl-2,3,4,5,6,7-hexahydro-1H-isoindol-1-one Example12a: ethyl6,6-dimethyl-2-(trifluoromethylsulfonyloxy)cyclohex-1-enecarboxylate

To a stirred suspension of sodium hydride (0.8 g, 20 mmol) in drydiethyl ether (30 mL) under an argon atmosphere at −20° C. was drop wiseadded ethyl 2,2-dimethyl-6-oxocyclohexanecarboxylate (1.98 g, 10 mmol).The mixture was stirred at −20° C. for 30 minutes and then at roomtemperature for 30 minutes. The mixture was cooled again to −20° C. andtrifluoromethansulfonic anhydride (2.5 ml, 15 mmol) was added drop wise.The reaction was stirred at −20° C. for 1 hour, and then warmedgradually to room temperature and stirred for 1 hour. The reaction wascooled to 0° C. and diethyl ether (20 mL) was added. Water (50 mL) wasadded slowly and the layers were separated. The aqueous layer wasextracted with diethyl ether (50 mL×3). The combined organic phase waswashed with saturated aqueous sodium bicarbonate (100 mL) and brine (100mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure. Purification of the residue by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=50/1) gave 2.6 gof a light yellow liquid that was repurified by silica gel columnchromatography (eluent: hexanes/ethyl acetate=1/0->500/1) to afford thetitle compound (900 mg, Yield: 27%). R_(f)=0.6 (20:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 4.26 (q, J=7.6 Hz, 2H),2.37 (t, J=6.8 Hz, 2H), 1.84-1.80 (m, 2H), 1.52-1.48 (m, 2H), 1.32 (t,J=7.2 Hz, 3H), 1.19 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 331 (M+H⁺).

Example 12b: ethyl 2-cyano-6,6-dimethylcyclohex-1-enecarboxylate

To a stirred solution of the product of Example 12a (802 mg, 2.43 mmol)in dimethylformamide (20 mL) was sequentially added zinc cyanide (426mg, 3.65 mmol), water (0.5 mL) andtetrakis(triphenylphosphine)palladium(0) (560 mg, 0.48 mmol). Thereaction was heated to 100° C. under argon for 5 hours. The reaction wascooled to room temperature and water (200 mL) was added. The mixture wasextracted with ethyl acetate (150 mL×4) and the combined organic phasewas washed with brine (200 mL×2), dried over anhydrous sodium sulfateand concentrated in vacuo. The crude product was purified by silica gelcolumn chromatography (eluent: petroleum ether/ethyl acetate=50/1->20/1)to afford the title compound as a colorless liquid (360 mg, Yield: 71%).R_(f)=0.3 (20:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃)δ 4.32 (q, J=7.2 Hz, 2H), 2.34-2.30 (m, 2H), 1.75-1.72 (m, 2H),1.53-1.50 (m, 2H), 1.36 (t, J=7.2 Hz, 3H), 1.19 (s, 6H) ppm; Massspectrum (ESI+ve) m/z 208 (M+H⁺).

Example 12: 7,7-dimethyl-2,3,4,5,6,7-hexahydro-1H-isoindol-1-one

To a stirred solution of the product of Example 12b (287 mg, 1.38 mmol)in THF (15 mL) was added Raney nickel (˜0.5 g). aqueous ammonia wasadded to make the reaction mixture become basic (pH-10, 8.5 ml). Thereaction mixture was stirred at room temperature under hydrogen for 2days. The reaction was filtered and the filtrate concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (eluent: dichloromethane/methanol=20/1->10/1) and thenrepurified by silica gel column chromatography (eluent: petroleumether/ethyl acetate=1/1) and then repurified again (eluent:dichloromethane/methanol=40/1) to give the title compound as whitecrystals (68 mg, Yield: 30%). Mp=186-188° C.; R_(f)=0.6 (20:1dichloromethane/Methanol); ¹H NMR (400 MHz, CDCl₃) δ 6.07 (bs, 1H), 3.75(s, 2H), 2.24-2.20 (m, 2H), 1.76-1.70 (m, 2H), 1.52-1.49 (m, 2H), 1.25(s, 6H) ppm; Mass spectrum (ESI+ve) m/z 166 (M+H⁺).

Example 13: 2,7,7-trimethyl-2,3,4,5,6,7-hexahydro-1H-isoindol-1-one

To a solution of the product of Example 12 (100 mg, 0.61 mmol) in drydichloromethane (10 mL) at 0° C. under argon was sequentially added1,8-bis(dimethylamino)naphthalene (1.44 g, 6.71 mmol) andtrimethyloxonium tetrafluoroborate (812 mg, 5.49 mmol) at 0° C. Thereaction was stirred at room temperature for 4 hours. The reactionmixture was concentrated under reduced pressure and the residue purifiedby prep-TLC to give the title compound as light pink solid (6 mg, Yield:5%). Mp=40-43° C.; R_(f)=0.5 (1:1 petroleum ether/ethyl acetate); ¹H NMR(400 MHz, CDCl₃) δ 3.69 (s, 2H), 2.98 (s, 3H), 2.20 (t, J=6.0 Hz, 2H),1.73-1.70 (m, 2H), 1.51-1.48 (m, 2H), 1.24 (s, 6H) ppm; Mass spectrum(ESI+ve) m/z 180 (M+H⁺).

Example 14:(±)-(3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneoxime

To a solution of the product of Example 1 (76.8 mg, 0.4 mmol) inpyridine (3.0 mL) was added hydroxylamine hydrochloride (294 mg, 4.0mmol) and the mixture was heated to reflux for 6 hours. The reaction wasconcentrated under reduced pressure and the residue purified by silicagel column chromatography to give the title compound as a white solid(58 mg, Yield: 70%). Mp=101-102° C.; R_(f)=0.6 (10:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 6.31 (s, 1H), 2.15 (s,1H), 1.84 (s, 3H), 1.84-1.81 (m, 1H), 1.70-1.49 (m, 1H), 1.41-1.48 (m,4H), 1.07 (s, 3 H), 1.06 (s, 3H), 0.76 (s, 3H) ppm; Mass spectrum(ESI+ve) m/z 208 (M+H⁺).

Example 15:(±)-(1S,3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol

To a solution of the product of Example 1 (100 mg, 0.52 mmol) in ethanol(5 mL) at 0° C. was added cerium trichloride heptahydrate (233 mg, 0.62mmol) and sodium borohydride (23 mg, 0.62 mmol). The mixture was warmedto room temperature stirred overnight. The reaction was quenched withwater and then the organics were extracted with ethyl acetate (20 mL×3).The combined organic phase was washed with water (20 mL) and brine (20mL), dried over anhydrous sodium sulfate and then concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to afford the title compound as a white solid (20 mg,Yield: 20%). Mp=55-56° C.; R_(f)=0.5 (10:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 5.25 (d, J=1.2 Hz, 1H), 4.55 (d,J=7.6 Hz, 1H), 1.62 (t, J=1.6 Hz, 3H), 1.59-1.45 (m, 1H), 1.40-1.23 (m,6H), 1.17 (s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 0.96-0.88 (m, 1H) ppm;Mass spectrum (ESI+ve) m/z 177 (M−H₂O+H⁺).

Example 16:(±)-(3R,3aS,7aS)-3,3a,7,7-tetramethyloctahydro-1H-inden-1-one oxime

To a solution of the product of Example 9 (77.6 mg, 0.4 mmol) inpyridine (3.0 mL) was added hydroxylamine hydrochloride (294 mg, 4.0mmol) and the mixture was heated to reflux for 6 hours. The reactionmixture was concentrated under reduced pressure and the residue waspurified by silica gel column chromatography to afford the titlecompound as a white solid (51 mg, Yield: 61%). Mp=144-145° C.; R_(f)=0.6(10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 6.89 (s,1H), 2.72 (dd, J=18.4, 8.0 Hz, 1H), 2.11-2.04 (m, 2H), 1.62-1.31 (m,4H), 1.39 (s, 3H), 1.38-1.17 (m, 5H), 1.13 (s, 3H), 1.10-0.98 (m, 1H),0.89 (d, J=6.8 Hz, 3H) ppm; Mass spectrum (ESI+ve) m/z 210 (M+H⁺).

Example 17: 7,7-dimethyloctahydro-1H-isoindol-1-one

To liquid ammonia (20 mL) at −78° C. was added lithium (˜20 mg). Thesolution became deep blue after stirring for 5 minutes. Then a solutionof the product of Example 12 (30 mg, 0.18 mmol) in tetrahydrofuran (1.5mL) was added slowly. The reaction mixture was stirred at −78° C. for 1hour and then warmed gradually to room temperature and stirred overnightto evaporate the ammonia. Saturated aqueous ammonium chloride (20 mL)was added and the resulting mixture was extracted with dichloromethane(15 mL×4). The combined organic phase was dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compoundas a light yellow solid (26 mg, Yield: 87%). Mp=141-148° C.; R_(f)=0.5(10:1 dichloromethane/ethyl acetate); ¹H NMR (400 MHz, CDCl₃+D₂O)(Major. trans) δ 3.28-3.24 (m, 1H), 2.90-2.84 (m, 1H), 2.12-2.03 (m,1H), 1.87-1.83 (m, 1H), 1.66-1.58 (m, 2H), 1.53-1.39 (m, 3H), 1.26 (s,3H), 1.19-1.11 (m, 1H), 0.97 (s, 3H); (Minor, cis) δ 3.28-3.24 (m,0.4H), 2.3-2.90 (m, 0.4H), 2.57-2.49 (m, 1H), 2.04 (d, J=7.6 Hz),1.87-1.83 (m, 1H), 1.66-1.58 (m, 0.8H), 1.53-1.39 (m, 0.8H), 1.26-1.11(m, 0.8 Hz), 1.18 (s, 1.4H), 1.04 (s, 1.5H) ppm; Mass spectrum (ESI+ve)m/z 168 (M+H⁺).

Example 18: (±)-(1S,3aS,7aR)-4,4,7a-trimethyloctahydro-1H-inden-1-ol

To a stirred solution of the product of Example 5 (50 mg, 0.28 mmol)cooled to 0° C. in methanol/tetrahydrofuran (0.5 mL/3.5 mL) was addedsodium borohydride (13 mg, 0.34 mmol). The reaction mixture was warmedto room temperature and stirred for 3 hours. The reaction was quenchedwith 5% hydrochloric acid (3 mL) and the organics were extracted withethyl acetate (50 mL). The organic layer was washed with brine (30mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to afford the title compound as a white solid (46 mg,Yield: 90%). Mp=59.7-60.8° C.; R_(f)=0.2 (20:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 3.66 (t, J=8.8 Hz, 1H), 2.02-1.97(m, 1H), 1.60-1.44 (m, 5H), 1.40-1.27 (m, 3H), 1.18-1.10 (m, 3H), 1.13(s, 3H), 1.04 (s, 3H), 0.80 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 82.8,52.1, 42.7, 34.1, 32.0, 30.5, 28.9, 28.4, 25.4, 24.6, 22.6, 18.4 ppm;Mass spectrum (ESI+ve) m/z 165 (M−H₂O+H⁺).

Example 19:(±)-(3aR,7aS)-3,3a,7,7-tetramethyl-3a,4,5,6,7,7a-hexahydro-O-methyloxime

Sodium hydride (9.6 mg, 0.24 mmol) was added to a solution of theproduct of Example 14 (41 mg, 0.2 mmol) in tetrahydrofuran (3 mL) at 0°C., and the resulting solution was allowed to warm to room temperatureand stirred for 2 hours. Then, methyl iodide (0.05 mL, 1.0 mmol) wasadded drop wise and the resulting mixture was stirred for 2 hours. Water(20 mL) was added and the mixture was extracted with ethyl acetate (60mL×2) and the combined organic phase was washed with brine (60 mL),dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto give the title compound as a colorless oil (20 mg, Yield: 45%).R_(f)=0.7 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃)δ 6.20 (s, 1H), 3.86 (s, 3H), 2.13 (s, 1H), 1.84 (s, 3H), 1.51-1.24 (m,6H), 1.10 (s, 3H), 1.05 (s, 3H), 0.75 (s, 3H) ppm; Mass spectrum(ESI+ve) m/z 222 (M+H⁺).

Example 20:(±)-(1R,7aS)-1,4,4,7a-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of the product of Example 6 (98 mg, 0.55 mmol) in drytetrahydrofuran (4.0 mL) at 0° C. was added lithium diisopropylamide(0.42 mL, 0.84 mmol) and then the mixture was allowed to warmed to roomtemperature the solution was stirred for 1 hour. The mixture was cooledto −78° C. and methyl iodide (0.27 mL, 5.5 mmol) was added. The solutionwas allowed to gradually warm to room temperature and stirred forovernight. The mixture was poured into saturated aqueous ammoniumchloride and then it was extracted with ethyl acetate (75 mL). Theorganic layer was washed by saturated aqueous sodium bicarbonate (50mL×2) and brine (50 mL×2), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to afford the title compound as a colorlessoil (70 mg, Yield: 76%). R_(f)=0.9 (20:1 petroleum ether/ethyl acetate);¹H NMR (400 MHz, CDCl₃) δ 5.81 (s, 1H), 2.16 (q, J=8.0 Hz, 1H),1.97-1.85 (m, 1H), 1.70-1.59 (m, 3H), 1.40-1.29 (m, 5H), 1.27 (s, 3H),1.20 (s, 3H), 1.05 (d, J=7.6 Hz, 3H) ppm; Mass spectrum (ESI+ve) m/z 193(M+H⁺).

Example 21:1,1,4,4,7a-pentamethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of the product of Example 20 (30 mg, 0.15 mmol) intetrahydrofuran (1 mL) 0° C. was added lithium diisopropylamide (0.14mL, 0.28 mmol) and then the mixture was allowed to warmed to roomtemperature and the solution was stirred for 1 hour. The mixture wascooled to −78° C. and methyl iodide (0.10 mL, 1.8 mmol) was added. Thesolution was allowed to gradually warm to room temperature stirred atroom temperature overnight. The mixture was poured into saturatedaqueous ammonium chloride and then the organics were extracted withethyl acetate (25 mL). The organic layer was washed with saturatedaqueous sodium bicarbonate (50 mL×2) and brine (50 mL×2), dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to afford thetitle compound as a yellow solid (20 mg, Yield: 64%). Mp=<15° C.;R_(f)=0.4 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl3)δ 5.80 (s, 1H), 1.90-1.86 (m, 1H), 1.65-1.60 (m, 3H), 1.48-1.34 (m, 2H),1.26 (s, 3H), 1.21 (s, 3H), 1.20 (s, 3H), 1.01 (s, 6H) ppm; Massspectrum (El+ve) m/z 206 (M⁺).

Example 22:(±)-((3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-2-yl)methanol

To a solution of the product of Example 8 (100 mg, 0.45 mmol) intetrahydrofuran (12 mL) at 0° C. was added lithium aluminum hydride (34mg, 0.90 mmol) and the mixture was stirred for 3 hours. The reaction wasquenched with and water and then the organics were extracted with ethylacetate (100 mL). The organic layer was washed with water (50 mL×2) andbrine (50 mL×2), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. Purification of the residue by silica gel columnchromatography gave the title compound as a colorless oil (68 mg, Yield:78%). R_(f)=0.2 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 5.44 (s, 1H), 4.13 (s, 2H), 2.22-2.17 (m, 2H), 1.67-1.62 (m,1H), 1.52-1.45 (m, 2H), 1.40-1.36 (m, 2H), 1.32-1.25 (m, 2H), 1.18 (s,3H), 1.17-1.09 (m, 1H), 1.02 (s, 3H), 0.83 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 139.9, 139.2, 62.4, 56.2, 45.1, 35.3, 35.2, 35.1, 32.0, 31.0,29.4, 26.8, 18.3 ppm; Mass spectrum (ESI+ve) m/z 177 (M−H₂O+H⁺).

Example 23:(±)-(3aS,7aS)-2-(methoxymethyl)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene

To a stirred solution of the product of Example 22 (54 mg, 0.28 mmol) indichloromethane (8 mL) at 0° C. was added1,8-bis(dimethylamino)naphthalene (600 mg, 2.8 mmol) andtrimethyloxonium tetrafluoroborate (414 mg, 2.8 mmol). The mixture waswarmed to room temperature and stirred for 4 hours. The reaction wasquenched with saturated aqueous sodium bicarbonate (20 mL) and thenextracted with dichloromethane (50 mL). The organic layer was washed by5% hydrochloric acid (30 mL×3) and brine (30 mL×2), dried over anhydroussodium sulfate and concentrated under reduced pressure. Purification ofthe residue by silica gel column chromatography afforded the titlecompound as a colorless oil (49 mg, Yield: 84%). R_(f)=0.5 (50:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 5.39 (s, 1H),3.83 (s, 2H), 3.25 (s, 3H), 2.14-2.11 (m, 2H), 1.58 (dd, J=10.0, 9.2 Hz,1H), 1.41-1.37 (m, 2H), 1.32-1.17 (m, 3H), 1.11 (s, 3H), 1.10-1.01 (m,1H), 0.94 (s, 3H), 0.81 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 140.4,136.0, 70.7, 57.0, 55.1, 44.2, 34.4, 34.3, 34.2, 30.9, 30.0, 28.4, 25.8,17.3 ppm; Mass spectrum (El+ve) m/z 208 (M)⁺.

Example 24:(±)-(1S,3aS,7aR)-1-methoxy-4,4,7a-trimethyloctahydro-1H-indene

To a stirred solution of the product of Example 18 (50 mg, 0.28 mmol)cooled to 0° C. in methanol/tetraydrofuran (0.5 mL/3.5 mL) was addedsodium borohydride (13 mg, 0.34 mmol). Then the solution was warmed toroom temperature and stirred for 3 hours. The reaction was quenched with5% hydrochloric acid (3 mL) and the mixture was extracted with ethylacetate (50 mL). The organic layer was washed with brine (30 mL×2),dried over anhydrous sodium sulfate and concentrated under reducedpressure. The to residue was purified by silica gel columnchromatography to give the title compound as a white solid (46 mg,Yield: 90%). Mp=59.7-60.8° C.; R_(f)=0.2 (20:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 3.66 (t, J=9.0 Hz, 1H), 2.02-1.97(m, 1H), 1.60-1.44 (m, 5H), 1.40-1.27 (m, 3H), 1.18-1.10 (m, 3H), 1.13(s, 3H), 1.04 (s, 3H), 0.80 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 82.8,52.1, 42.7, 34.1, 32.0, 30.5, 28.9, 28.4, 25.4, 24.6, 22.6, 18.4 ppm;Mass spectrum (ESI+ve) m/z 165 (M−H₂O+H⁺).

Example 25: (±)-(3aS,7aS)-methyl3a,7,7-trimethyloctahydro-1H-indene-2-carboxylate

To a solution of the product of Example 8 (78 mg, 0.35 mmol) in methanol(8 mL) was added Pd/C (˜50 mg). Then the mixture was stirred under anatmosphere of hydrogen overnight. The reaction mixture was filtered andthe filtrate concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to give the title compoundas a colorless oil (55 mg, Yield: 71%). R_(f)=0.6 (100:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 3.66 (s, 3H), 2.78-2.74(m, 1H), 1.96 (t, J=9.6 Hz, 2H), 1.77 (d, J=9.6 Hz, 2H), 1.55-1.35 (m,5H), 1.20-1.15 (m, 2H), 1.11 (s, 3H), 1.02 (s, 3H), 0.84 (s, 3H); ¹³CNMR (100 MHz, CDCl₃) δ 177.70, 55.60, 51.68, 45.96, 40.54, 39.49, 34.34,33.97, 32.78, 31.99, 31.63, 28.89, 27.87, 19.39 ppm; Mass spectrum(ESI+ve) m/z 225 (M+H⁺).

Example 26:(±)-((3a5,7aS)-3a,7,7-trimethyloctahydro-1H-inden-2-yl)methanol

To a solution of the product of Example 25 (70 mg, 0.31 mmol) intetrahydrofuran (8 mL) at 0° C. was added lithium aluminum hydride (24mg, 0.62 mmol). The mixture was stirred for 3 hours and then thereaction was quenched with water. The organics were extracted with ethylacetate, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to afford the title compound as a colorless oil (54 mg,Yield: 89%). R_(f)=0.4 (5:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 3.58-3.50 (m, 2H), 2.18-2.10 (m, 1H), 1.84-1.80 (m, 1H),1.67 (t, J=12.4 Hz, 1H), 1.56-1.49 (m, 1H), 1.40-1.23 (m, 6H), 1.21-1.12(m, 3H), 1.12 (s, 3H), 1.03 (s, 3H), 0.84 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 68.8, 55.6, 46.1, 40.2, 38.1, 35.7, 34.7, 32.9, 32.0, 31.9,29.1, 28.6, 19.6 ppm; Mass spectrum (ESI+ve) m/z 179 (M−H₂O+H⁺).

Example 27: 7,7-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one Example27a: 2-(hydroxymethylene)-6-methylcyclohexanone

To an ice cold suspension of powdered sodium methoxide (24.5 g, 454mmol) in toluene (450 mL) was added 2-methylcyclohexanone (60 g, 178mmol) and ethyl formate (79.2 g, 1069 mmol). The mixture was stirred atroom temperature overnight. Ice water and toluene were added and thephases were separated. The organic phase was washed with 10% sodiumhydroxide (100 mL×2). The aqueous layer was acidified with dilutehydrochloric acid to pH 3 and then extracted with diethyl ether (200mL×3). The combined organic layer was washed with water (100 mL×2) andbrine (100 mL×2) and dried over anhydrous sodium sulfate. Concentrationunder reduced pressure gave the title compound as a light orange oil (50g, Yield: 66%).

Example 27b: 2-(isopropoxymethylene)-6-methylcyclohexanone

To a solution of the product of Example 27a (50 g, 357 mmol) in acetone(500 mL) was added potassium carbonate (74.1 g, 536 mmol) and2-iodopropane (45 mL, 446 mmol) and the reaction mixture was refluxedovernight. After concentration under reduced pressure and the residuewas extracted with ether (800 mL) and the organic layer was washed with5% aqueous sodium hydroxide (100 mL×2), brine (100 mL), dried overanhydrous sodium sulfate, and concentrated under reduced pressure togive the title compound as a light yellow oil (55 g, Yield: 84%), whichwas used in the next step without any further purification.

Example 27c: (E)-6-(tert-butoxymethylene)-2,2-dimethylcyclohexanone and(E)-6-(isopropoxymethylene)-2,2-dimethylcyclohexanone

To a solution of potassium tert-butoxide (106 g, 945 mmol) intetrahydrofuran (550 mL) cooled to 0° C. was added the product ofExample 27b (55 g, 302 mmol). The mixture was stirred at 0° C. for 10minutes and then methyl iodide (141 g, 993 mmol) was added. The mixturebegan to reflux and when reflux ceased the cooling bath was removed andthe mixture was then stirred at room temperature for 1 hour. The mixturewas filtered and the filtrate concentrated under reduced pressure. Theresidue was diluted with diethyl ether (600 mL), washed by 10% aqueoussodium hydroxide (100 mL×2), brine (100 mL), dried over anhydrous sodiumsulfate and concentrated under reduced pressure to give 45 g of anorange oil. Purification of 6.0 g of material by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=1/0->30/1 gave a4:1 mixture of (E)-6-(tert-butoxymethylene)-2,2-dimethylcyclohexanoneand (E)-6-(isopropoxymethylene)-2,2-dimethylcyclohexanone as a colorlessoil (3.52 g)¹H NMR (400 MHz, CDCl₃) Major: δ 7.58 (t, J=2.0 Hz, 1H),2.41-2.39 (m, 2H), 1.70-1.66 (m, 4H), 1.35 (s, 9H), 1.12 (s, 6H); Minor:δ 7.37 (t, J=1.8 Hz, 1H), 4.23-4.15 (m, 1H), 2.41-2.39 (m, 2H),1.70-1.66 (m, 4H), 1.29 (d, J=6.4 Hz, 6H), 1.12 (s, 6H).

Example 27d: 2,2-dimethylcyclohexanone

To the product of Example 27c (3.52 g, 16.7 mmol) was added a solutionof 20% aqueous sodium hydroxide (28 mL). The resulting mixture washeated to reflux for 24 hours then ethanol (20 mL) was added andrefluxing was continued for additional 45 minutes. The mixture wasdiluted with water (50 mL) and then it was extracted with petroleumether (50 mL×4).

The combined organic phase was washed with brine (100 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure to givethe title compound as light yellow liquid (0.99 g, Yield: 47%). ¹H NMR(400 MHz, CDCl₃) δ 2.39 (t, J=6.8 Hz, 2H), 1.85-1.80 (m, 2H), 1.75-1.71(m, 2H), 1.67-1.62 (m, 2H), 1.11 (s, 6H) ppm.

Example 27e: 6-allyl-2,2-dimethylcyclohexanone

Potassium t-butoxide (393 mg, 3.5 mmol) was added to t-butanol (5 mL) inone portion at room temperature. The mixture was stirred for 10 minutesunder argon. A solution of the product of Example 27d (441 mg, 3.5 mmol)in t-butanol (2 mL) was added slowly. After stirring for 10 minutes,allyl bromide (0.46 ml, 5.25 mmol) was added and the mixture was stirredat room temperature overnight. Another portion of potassium t-butoxide(39 mg, 0.35 mmol) was added and the reaction was heated to reflux for 1hour. The mixture was partitioned between diethyl ether (30 mL) andwater (30 mL). The aqueous layer was extracted with diethyl ether (30mL×2) and the combined organic phase were dried over anhydrous sodiumsulfate and concentrated under reduced pressure. Purification of theresidue by silica gel column chromatography afforded the title compoundas a colorless oil contaminated with2,2-diallyl-6,6-dimethylcyclohexanone (256 mg). This material was useddirectly for the next step without further purification. ¹H NMR (400MHz, CDCl₃) Major: δ 5.83-5.73 (m, 1H), 5.04-4.97 (m, 2H), 2.64-2.49 (m,2H), 2.15-2.09 (m, 1H), 1.96-1.65 (m, 5H), 1.29-1.25 (m, 1H), 1.19 (s,3H), 1.05 (s, 3H); Minor: δ 5.67-5.57 (m, 2H), 5.04-4.97 (m, 4H),2.41-2.32 (m, 2H), 2.24-2.18 (m, 2H), 1.96-1.54 (m, 6H), 1.10 (s, 6H)ppm.

Example 27f: 2,2-dimethyl-6-(2-oxopropyl)cyclohexanone

A 50 ml round-bottom-flask was charged with palladium dichloride (14 mg,0.076 mmol), cupric acetate hydrate (76 mg, 0.38 mmol),dimethylacetamide (3.5 mL) and water (0.5 mL) and then the product ofExample 27e (251 mg, 1.51 mmol) was added to the mixture. The system wascooled to −78° C. then evacuated with vacuum and back-filled with anatmosphere of oxygen. The mixture was warmed to room temperature andstirred vigorously for 60 hours under oxygen. The reaction was thendirectly loaded onto a silica gel column and purified by flashchromatography (eluent: petroleum ether/ethyl acetate=1/0->100/1) toafford the title compound as a light yellow liquid (101 mg, Yield: 37%).¹H NMR (400 MHz, CDCl₃) δ 3.28-3.20 (m, 1H), 2.93 (dd, J=17.6, 7.6 Hz,1H), 2.21 (s, 3H), 2.11 (dd, J=17.2, 4.8 Hz, 1H), 2.06-2.03 (m, 1H),1.98-1.87 (m, 1H), 1.82-1.76 (m, 1H), 1.70-1.64 (m, 1H), 1.59-1.50 (m,1H), 1.39-1.31 (m, 1H), 1.23 (s, 3H), 1.03 (s, 3H) ppm; Mass spectrum(ESI+ve) m/z 205 (M+Na⁺).

Example 27: 7,7-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a stirred solution of the product of Example 27f (101 mg, 0.55 mmol)in xylenes (3 mL) was added freshly powdered potassium hydroxide (14 mg,0.25 mmol). The reaction was stirred at 120° C. for 3 hours. Thereaction solution was directly loaded onto a silica gel column andpurified by silica gel flash chromatography (eluent: petroleumether/ethyl acetate=20/1) to give a light yellow oil which was furtherpurified by prep-HPLC to obtain a colorless oil which was furtherpurified again by silica gel flash chromatography (eluent: petroleumether/ethyl acetate=10/1) to afford the title compound as a colorlessoil (5 mg, Yield: 5%). R_(f)=0.15 (10:1 petroleum ether/ethyl acetate);¹H NMR (400 MHz, CDCl₃) δ 5.86 (s, 1H), 2.77 (dd, J=14.0, 3.6 Hz, 1H),2.55 (d, J=6.4 Hz, 1H), 2.32 (dd, J=19.0, 6.6 Hz, 1H), 2.25-2.15 (m,2H), 1.85-1.81 (m, 1H), 1.68-1.62 (m, 1H), 1.54-1.44 (m, 2H), 1.02 (s,3H), 0.69 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 165 (M+H⁺).

Example 28:(±)-(3aR,7aS)-3a,7,7-trimethyl-3-phenyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 28a: 1-(2,6,6-trimethylcyclohex-1-enyl)ethanol

To a stirred solution of 2,6,6-trimethylcyclohex-1-enecarbaldehyde (950mg, 6.24 mmol) in dry tetrahydrofuran (25 mL) at −78° C. under argon wasadded methyl magnesium iodide (3.0 M, 5.2 mL, 15.6 mmol). The resultingsolution was warmed gradually to room temperature and stirred for 1hour. Additional dry tetrahydrofuran (15 mL) was added and the reactionwas stirred overnight. Saturated aqueous ammonium chloride (25 mL) wasadded to quench the reaction and the mixture was diluted with water (25mL) and the organics were extracted with ethyl acetate (50 mL×3). Thecombined organic phase was washed with brine (50 mL×2), dried overanhydrous sodium sulfate and concentrated under reduced pressure.Purification of the residue by silica gel flash chromatography gave thetitle compound as a colorless liquid (856 mg, Yield: 82%). ¹H NMR (400MHz, CDCl₃) δ 4.51 (q, J=6.4 Hz, 1H), 1.98-1.91 (m, 2H), 1.86 (s, 3H),1.56-1.52 (m, 2H), 1.44 (d, J=6.4 Hz, 3H), 1.45-1.35 (m, 3H), 1.09 (s,3H), 0.96 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 151 (M−H₂O+H⁺).

Example 28b: 1-(2,6,6-trimethylcyclohex-1-enyl)ethanone

To a stirred solution of the product of Example 28a (850 mg, 5.06 mmol)in dichloromethane (25 mL) at 0° C. was added Dess-Martin periodinane(3.88 g, 9.14 mmol). The reaction was stirred at room temperature for1.5 hours. The reaction mixture was concentrated and then the residuediluted with petroleum ether (100 mL). The mixture was filtered and thefiltrate concentrated under reduced pressure. Purification of theresidue by silica gel column chromatography (eluent: petroleumether/ethyl acetate=100/1) gave a colorless liquid that was furtherpurified by silica gel column chromatography (eluent: petroleumether/ethyl acetate=100/1->25/1) to afford the title compound as acolorless liquid (87 mg, Yield: 10%). R_(f)=0.5 (20:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃+D₂O) δ 2.28 (s, 3H), 1.95(t, J=6.4 Hz, 2H), 1.67-1.64 (m, 2H), 1.58 (s, 3H), 1.45-1.42 (m, 2H),1.07 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 189 (M+Na⁺).

Example 28c:3-hydroxy-3-phenyl-1-(2,6,6-trimethylcyclohex-1-enyl)propan-1-one

To a stirred solution of the product of Example 28b (100 mg, 0.60 mmol)in dry tetrahydrofuran (1.5 mL) at −78° C. was slowly added lithiumdiisopropylamide (0.36 mL, 0.72 mmol). After 15 minutes, a solution ofbenzaldehyde (76 mg, 0.72 mmol) in dry tetrahydrofuran (1.5 mL) wasadded. The mixture was stirred at −78° C. for 30 minutes. The mixturewas quenched with saturated aqueous ammonium chloride and the mixturewas extracted with diethyl ether (30 mL×3). The combined organic phasewas washed with brine (30 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=100/1) to give a colorless oil which was further purified bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=250/1->50/1) to afford the title compound as a pale yellow solid(90 mg, Yield: 55%). R_(f)=0.2 (20:1 petroleum ether/ethyl acetate); ¹HNMR (400 MHz, CDCl₃+D₂O) δ 7.39-7.33 (m, 4H), 7.27-7.26 (m, 1H), 5.22(t, J=6.0 Hz, 1H), 2.94 (d, J=6.0 Hz, 2H), 1.95 (t, J=6.4 Hz, 2H),1.68-1.62 (m, 2H), 1.58 (s, 3H), 1.44-1.41 (m, 2H), 1.08 (s, 3H), 1.07(s, 3H) ppm.

Example 28d:(E)-3-phenyl-1-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

A solution of the product of Example 28c (29 mg, 0.11 mmol) inphosphoric acid (2 mL) and tetrahydrofuran (5 mL) was stirred at roomtemperature for 22 hours. The mixture was added to saturated aqueoussodium bicarbonate (30 mL). The resulting mixture was extracted withethyl acetate (30 mL×3). The combined organic phase was washed withbrine (30 mL), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. Purification of the residue by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=100/1) affordedthe title compound as a yellow oil (29 mg, Yield: 100%). R_(f)=0.6 (10:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.57-7.55 (m,2H), 7.43-7.39 (m, 3H), 7.41 (d, J=16.0 Hz, 1H), 6.76 (d, J=16.0 Hz,1H), 2.05 (t, J=6.4 Hz, 2H), 1.77-1.71 (m, 2H), 1.58 (s, 3H), 1.54-1.51(m, 2H), 1.08 (s, 6H) ppm.

Example 28:(±)-(3aR,7aS)-3a,7,7-trimethyl-3-phenyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

A solution of the product of Example 28d (23 mg, 0.09 mmol) inmethansulfonic acid (2 ml) was stirred at 50° C. for 3 hours. Themixture was added to saturated aqueous sodium bicarbonate (30 mL). Theresulting material was extracted with ethyl acetate (30 mL×3). Thecombined organic phase was washed with brine (30 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure.Purification by prep-TLC (eluent: petroleum ether/ethyl acetate=30/1)afforded the title compound as a white solid (7 mg, Yield: 30%).Mp=46-48° C.; R_(f)=0.5 (10:1 petroleum ether/ethyl acetate); ¹H NMR(400 MHz, CDCl₃+D₂O) δ 7.41 (br, m, 5H), 6.08 (s, 1H), 2.03 (s, 1H),2.00-1.95 (m, 1H), 1.72-1.49 (m, 3H), 1.46-1.33 (m, 2H), 1.33 (s, 3H),1.22 (s, 3H), 0.97 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 255 (M+H⁺).

Example 29:(±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 29a:3-hydroxy-4-methyl-1-(2,6,6-trimethylcyclohex-1-enyl)pentan-1-one

A stirred solution of the product of Example 28b (86 mg, 0.52 mmol) indry tetrahydrofuran (2 mL) at −78° C. was slowly added lithiumdiisopropylamide (0.39 mL, 0.78 mmol). After 15 minutes, a solution ofisobutyraldehyde (75 mg, 1.04 mmol) in dry tetrahydrofuran (2 mL) wasadded. The mixture was stirred at −78° C. for 2 hours and then themixture was quenched with saturated aqueous ammonium chloride. Themixture was extracted with diethyl ether (30 mL×3). The combined organicphase was washed with brine (30 mL), dried over anhydrous sodium sulfateand concentrated under reduced pressure. Purification of the residue bycolumn chromatography (eluent: petroleum ether/ethylacetate=100/1->25/1) gave the title compound as a colorless oil (99 mg,Yield: 80%). R_(f)=0.4 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 3.90-3.86 (m, 1H), 3.38 (d, J=2.4 Hz, 1H), 2.74 (d, J=18.4Hz, 1H), 2.57 (dd, J=18.4, 6.0 Hz, 1H), 1.96 (t, J=6.4 Hz, 2H),1.74-1.63 (m, 3H), 1.59 (s, 3H), 1.46-1.40 (m, 2H), 1.08 (s, 3H), 1.07(s, 3H), 0.95 (d, J=6.8 Hz, 3H), 0.92 (d, J=6.8 Hz, 3H) ppm.

Example 29:(±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

A solution of the product of Example 29a (84 mg, 0.35 mmol) inphosphoric acid (3 mL) was stirred at room temperature for 3 hours. Themixture was added to saturated aqueous sodium bicarbonate and themixture was extracted with ethyl acetate (30 mL×3). The combined organicphase was washed with brine (30 mL×2), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. Purification of theresidue by prep-TLC (eluent: petroleum ether/ethyl acetate=20/1 affordedthe title compound as a colorless oil (15 mg, Yield: 19%). R_(f)=0.5(10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃+D₂O) δ 5.86(s, 1H), 2.62-2.56 (m, 1H), 1.85 (s, 1H), 1.77-1.50 (m, 4H), 1.37-1.35(m, 2H), 1.19 (s, 6H), 1.16 (d, J=6.8 Hz, 3H), 1.15 (d, J=6.4 Hz, 3H),0.88 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 221 (M+H⁺).

Example 30: 4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-oneoxime

To a solution of the product of Example 6 (71 mg, 0.4 mmol) in pyridine(3.0 mL) was added hydroxylamine hydrochloride (278 mg, 4.0 mmol) andthe mixture was heated to reflux overnight. The reaction wasconcentrated under reduced pressure and the residue was purified bysilica gel column chromatography to afford the title compound as a whitesolid (54 mg, Yield: 75% yield). Mp=129-130° C.; R_(f)=0.3 (5:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃+D₂O) δ 5.84 (s,1H), 2.69 (d, J=18.0 Hz, 1H), 2.45 (d, J=18.4 Hz, 1H), 1.87-1.78 (m,2H), 1.58-1.53 (m, 2H), 1.34-1.24 (m, 5H), 1.17 (s, 6H) ppm; Massspectrum (ESI+ve) m/z 194 (M+H⁺).

Example 31: 4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-oneO-methyl oxime

To a solution of the product of Example 30 (39.8 mg, 0.21 mmol) intetrahydrofuran (5.0 mL) at 0° C. was added sodium hydride (10 mg, 0.25mmol) and the mixture was warmed to room temperature and stirred for 2hours. Methyl iodide (0.064 ml, 1.03 mmol) was added drop wise and thereaction was stirred overnight. Water was added to the mixture and thenthe organics were extracted with ethyl acetate. The combined organicphase was washed with brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to afford the title compound as a colorlessoil (6.9 mg, Yield: 15%). R_(f)=0.3 (50:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃+D₂O) δ 5.84 (s, 1H), 3.86 (s, 3H), 2.61(d, J=18.0 Hz, 1H), 2.39 (d, J=18.0 Hz, 1H), 1.84-1.77 (m, 2H),1.55-1.52 (m, 2H), 1.32-1.25 (m, 5H), 1.21 (s, 6H) ppm; Mass spectrum(ESI+ve) m/z 208 (M+H⁺).

Example 32: (±)-(3R,3aS,7aS)-3,3a,7,7-tetramethyloctahydro-1H-inden-1-ol

To a solution of the product of Example 9 (80 mg, 0.41 mmol) in methanol(5 mL) was added sodium borohydride (30 mg, 0.49 mmol) and ceriumtrichloride heptahydrate (184 mg, 0.49 mmol). The reaction was stirredat room temperature for 2 days. The reaction mixture was concentratedunder reduced pressure and then water (2 mL) was added. The mixture wasthen extracted with ethyl acetate (30 mL×3). The combined organic phasewas washed with water (20 mL) and brine (20 mL), dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound as a white solid (20 mg, Yield: 25%). Mp=98-102° C.; R_(f)=0.4(10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃+D₂O) δ 4.12(t, J=6.8 Hz, 1H), 1.88-1.41 (m, 5H), 1.25-1.13 (m, 4H), 1.06-1.00 (m,9H), 0.91-0.84 (m, 1H), 0.77 (d, J=6.8 Hz, 3H) ppm; Mass spectrum(ESI+ve) m/z 179 (M−H₂O+H⁺).

Example 33: (±)-(3aS,7aR)-4,4,7a-trimethyloctahydro-1H-inden-1-one oxime

To a solution of the product of Example 5 (50 mg, 0.28 mmol) in ethanol(5 mL) was added hydroxylamine hydrochloride (39 mg, 0.56 mmol) andpyridine (44 mg, 0.56 mmol). The mixture was heated to reflux for 2hours. The reaction mixture was concentrated under reduced pressure andthe residue diluted with ethyl acetate (50 mL). The organic phase waswashed with 1N hydrochloric acid (30 mL×3) and brine (30 mL×2), driedover anhydrous sodium sulfate and concentrated under reduced pressure.The residue was purified by silica gel column chromatography to affordthe title compound as a white solid (28 mg, Yield: 51%). Mp=117.6-118.6°C.; R_(f)=0.3 (30:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 8.38 (br, 1H), 2.64 (dd, J=19.0, 8.6 Hz, 1H), 2.38-2.29 (m,1H), 1.83-1.80 (m, 1H), 1.66-1.43 (m, 5H), 1.38-1.17 (m, 3H), 1.27 (s,3H), 1.04 (s, 3H), 0.84 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.8, 55.0,44.3, 35.4, 32.4, 31.8, 29.6, 29.5, 25.4, 24.7, 23.5, 18.7 ppm; Massspectrum (ESI+ve) m/z 196 (M+H⁺).

Example 34:(±)-(3aR,7aS)-3-ethyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 34a: (E)-1-(2,6,6-trimethylcyclohex-1-enyl)pent-2-en-1-one

To stirred solution of the product of 28b (140 mg, 0.84 mmol) in drytetrahydrofuran (2 mL) at −78° C. was slowly added lithiumdiisopropylamide (2M, 0.56 ml, 1.12 mmol). After 15 minutes, a solutionof propionaldehyde (98 mg, 1.68 mmol) in dry tetrahydrofuran (2 mL) wasadded. The mixture was stirred at −78° C. for 2 hours and then themixture was stirred room temperature overnight. The reaction wasquenched with saturated aqueous ammonium chloride and the mixture wasextracted with ethyl acetate (50 mL×3). The combined organic phase waswashed with brine (30 mL×2), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=200/1) gave the title compound as a pale yellow oil (98 mg,Yield: 57%). R_(f)=0.6 (20:1 petroleum ether/ethyl acetate; ¹H NMR (400MHz, CDCl₃) δ 6.78-6.72 (m, 1H), 6.11 (d, J=16.0 Hz, 1H), 2.29-2.22 (m,2H), 2.01-1.95 (m, 2H), 1.72-1.67 (m, 2H), 1.51 (s, 3H), 1.48-1.44 (m,2H), 1.08 (t, J=7.2 Hz, 3H), 1.02 (s, 6H).

Example 34:(±)-(3aR,7aS)-3-ethyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

A solution of the product of Example 34a (50 mg, 0.24 mmol) inphosphoric acid (2 mL) was stirred at room temperature for 4 hours. Themixture was added to saturated aqueous sodium bicarbonate (30 mL). Themixture was extracted with ethyl acetate (30 mL×3) and the combinedorganic phase was washed with brine (30 mL×2), dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by prep-TLC (eluent: petroleum ether/ethyl acetate=25/1) toafford the title compound as a pale yellow oil (26 mg, Yield: 53%).R_(f)=0.6 (10:1 petroleum ether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ5.81 (s, 1H), 2.33-2.26 (m, 2H), 1.86 (s, 1H), 1.71-1.61 (m, 2H),1.57-1.48 (m, 2H), 1.40-1.32 (m, 2H), 1.20 (s, 3H), 1.18 (s, 3H), 1.18(t, J=7.2 Hz, 3H), 0.89 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 207(M+H⁺).

Example 35:(±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 35a: 1-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-ol

To a solution of 2,6,6-trimethylcyclohex-1-enecarbaldehyde (400 mg, 2.63mmol) in dry tetrahydrofuran (8 mL) at room temperature under argon wasadded vinyl magnesium bromide (4.51 mL, 3.16 mmol). The mixture wasstirred for 10 minutes and then saturated aqueous ammonium chloride wasadded. The heterogeneous mixture was stirred for 5 min and thenextracted with diethyl ether (100 mL). The organic layer was washed withbrine (50 mL×2), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. Purification of the residue by silica gel columnchromatography gave the title compound as a colorless oil (360 mg,Yield: 76%). R_(f)=0.3 (20:1 petroleum ether/ethyl acetate; ¹H NMR (400MHz, CDCl₃) δ 6.11-6.03 (m, 1H), 5.24 (d, J=17.6 Hz, 1H), 5.11 (d,J=10.4 Hz, 1H), 4.82 (s, 1H), 1.95 (t, J=6.0 Hz, 2H), 1.75 (s, 3H),159-1.56 (m, 3H), 1.44 (t, J=6.0 Hz, 2H), 1.12 (s, 3H), 0.98 (s, 3H).

Example 35b: 1-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

To a solution of the product of Example 35a (355 mg, 1.97 mmol) inpentane (7 mL) was added manganese dioxide (2.57 g, 29.55 mmol). Thereaction was stirred under argon at room temperature for 12 h ours. Themixture was filtered and the filtrate concentrated to give a yellow oilthat was dissolved in dichloromethane (5 mL) at 0° C. and Dess-Martinperiodinane (835 mg, 1.97 mmol) was added. The resulting mixture wasstirred at room temperature for 1.5 hours. Water was added and themixture was extracted with dichloromethane (100 mL). The organic phasewas washed by saturated aqueous sodium bicarbonate (50 mL×2) and brine(50 mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure. Purification of the residue by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=50/1) afforded thetitle compound as a light yellow oil (242 mg, Yield: 69%). R_(f)=0.5(30:1 petroleum ether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ 6.38 (dd,J=17.6, 10.4 Hz, 1H), 6.13 (d, J=17.6 Hz, 1H), 5.97 (d, J=10.4 Hz, 1H),2.01 (t, J=6.4 Hz, 2H), 1.72-1.68 (m, 2H), 1.52 (s, 3H), 1.49-1.46 (t,J=6.0 Hz, 2H), 1.03 (s, 6H).

Example 35:(±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

A mixture of the product of Example 35b (230 mg, 1.29 mmol), phosphoricacid (500 mg) and formic acid (98%, 1.5 g) was kept at 80° C. for 1 hourunder argon. After cooling, the mixture was added to water (10 mL) andthe resulting solution was extracted with diethyl ether (100 mL). Theorganic layer was washed with 10% aqueous sodium carbonate (50 mL×2),water (50 mL×2) and brine (50 mL×2), dried over anhydrous sodium sulfateand concentrated in vacuo. The residue was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=50/1) to affordthe title compound as a light yellow syrup. (128 mg, Yield: 56%).R_(f)=0.5 (30:1 petroleum ether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ7.37 (d, J=5.6 Hz, 1H), 5.98 (d, J=5.6 Hz, 1H), 1.80 (s, 1H), 1.67-1.65(m, 1H), 1.60-1.57 (m, 3H), 1.37-1.35 (m, 2H), 1.20 (s, 6H), 0.95 (s,3H) ppm; Mass spectrum (ESI+ve) m/z 179 (M+H⁺).

Example 36: (±)-(1S,3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-H-inden-1-01

To a solution of the product of Example 35 (111 mg, 0.62 mmol) intetrahydrofuran (4 mL) was slowly added 9-borabicyclo[3.3.1]nonane (2.48mL, 1.24 mmol). The reaction mixture was stirred under argon at roomtemperature for 25 minutes, then methanol (1.5 mL) was slowly added andstirring was continued for 1 hour. Concentration of the reaction invacuo and purification of the residue by silica column chromatographyafforded the title compound as a white solid (28 mg, Yield: 25%).Mp=55.6-57.2° C.; R_(f)=0.3 (20:1 petroleum ether/ethyl acetate; ¹H NMR(400 MHz, CDCl₃) δ 5.68 (d, J=5.6 Hz, 1H), 5.56 (d, J=5.6 Hz, 1H), 4.68(br, 1H), 1.54-1.50 (m, 2H), 1.41-1.28 (m, 5H), 1.22 (s, 3H), 1.10 (s,3H), 1.06 (s, 3H), 0.99-0.93 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 143.6,127.9, 77.4, 63.0, 43.9, 35.0, 34.3, 29.6, 29.5, 27.8, 25.5, 15.6 ppm;Mass spectrum (ESI+ve) m/z 163 (M−H₂O+H⁺).

Example 37:(±)-(1R,3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol

To a solution of the product of Example 35 (77 mg, 0.43 mmol) intetrahydrofuran (4 mL) was slowly added 9-borabicyclo[3.3.1]nonane (2.58mL, 1.29 mmol). The reaction mixture was stirred under argon at roomtemperature for 2 hours and then methanol (1.5 mL) was slowly added andstirring was continued for 1 hour. The reaction mixture was concentratedin vacuo and the residue was purified by silica gel columnchromatography to afford the title compound as a colorless oil (15 mg,Yield: 20%). R_(f)=0.4 (20:1 petroleum ether/ethyl acetate; ¹H NMR (400MHz, CDCl₃) δ 5.94 (d, J=5.2 Hz, 1H), 5.83 (br, 1H), 4.62 (br, 1H),1.73-1.53 (m, 4H), 1.30-1.18 (m, 3H), 1.22 (s, 3H), 1.18 (s, 3H), 1.07(s, 3H), 0.86 (br, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 150.9, 129.5, 79.5,57.8, 45.3, 39.1, 37.5, 32.7, 31.2, 28.9, 26.3, 19.6 ppm; Mass spectrum(ESI+ve) m/z 163 (M−H₂O+H⁺).

Example 38:(±)-(1S,2R,3aS,7aR)-2-(hydroxymethyl)-4,4,7a-trimethyloctahydro-1H-inden-1-ol

To a solution of the product of Example 8b (50 mg, 0.21 mmol) intetrahydrofuran (5 mL) at 0° C. was added lithium aluminum hydride (16mg, 0.42 mmol) and the mixture was stirred for 3 hours. The reaction wasquenched with ethyl acetate and water. The mixture was filtered andconcentrated in vacuo. Purification of the residue by silica gel columnchromatography gave the title compound as a white solid (35 mg, Yield:78%). Mp=78.9-80.2° C.; R_(f)=0.4 (2:1 petroleum ether/ethyl acetate; ¹HNMR (400 MHz, CDCl₃) δ 3.94 (dd, J=10.0, 5.2 Hz, 1H), 3.78-3.76 (m, 1H),3.70-3.66 (m, 1H), 2.69 (t, J=5.2 Hz, 1H), 2.34-2.30 (m, 1H), 2.19 (d,J=5.2 Hz, 1H), 1.67-1.50 (m, 3H), 1.40-1.29 (m, 4H), 1.19-1.15 (m, 2H),1.16 (s, 3H), 1.04 (s, 3H), 0.81 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ84.6, 64.8, 52.0, 43.9, 40.4, 34.2, 31.7, 31.3, 28.7, 28.6, 26.340,26.267, 18.7 ppm; Mass spectrum (ESI+ve) m/z 213 (M+H⁺).

Example 39:(±)-(1S,2S,3aS,7aR)-2-(hydroxymethyl)-4,4,7a-trimethyloctahydro-1H-inden-1-ol

To a solution of the product of Example 8c (50 mg, 0.21 mmol) intetrahydrofuran (5 mL) at 0° C. was added lithium aluminum hydride (16mg, 0.42 mmol) and the mixture was stirred for 3 hours. The reaction wasquenched with ethyl acetate and water. The mixture was filtered andconcentrated in vacuo. Purification of the residue by silica gel columnchromatography gave the title compound as a white solid (40.9 mg, Yield:92%). Mp=103.6-105.4° C.; R_(f)=0.2 (2:1 petroleum ether/ethyl acetate;¹H NMR (400 MHz, CDCl₃) δ 3.76 (dd, J=9.4, 5.8 Hz, 1H), 3.63-3.58 (m,1H), 3.41 (d, J=8.8 Hz, 1H), 2.05-1.98 (m, 3H), 1.77-1.71 (m, 1H),1.55-1.51 (m, 2H), 1.34-1.25 (m, 3H), 1.18-1.13 (m, 3H), 1.13 (s, 3H),1.02 (s, 3H), 0.78 (s, 3H); ¹³C NMR (100 MHz, CDCl3) δ 86.8, 67.4, 50.9,43.6, 43.3, 34.0, 31.9, 30.4, 28.4, 26.5, 26.2, 24.6, 18.4 ppm; Massspectrum (ESI+ve) m/z 213 (M+H^(f)).

Example 40: (±)-(3aS,7aS)-3a,7,7-trimethylhexahydro-1H-inden-2(3H)-one

The solution of the product of Example 6 (50 mg, 0.28 mmol) in methanol(1 mL) was added dry Pd/C (10.0 mg) and the mixture was stirred under anatmosphere of hydrogen for 24 hours. The reaction was suction filteredand the filtrate concentrated under reduced pressure to give the titlecompound as a yellow semisolid (46.2 mg, Yield: 92%). R_(f)=0.4 (10:1petroleum ether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ 2.25 (m, 2H),2.10 (s, 2H), 1.80 (m, 1H), 1.59 (m, 1H), 1.49 (m, 1H), 1.34-1.30 (m,7H), 1.10 (s, 3H), 0.83 (s, 3H) ppm; Mass spectrum (El+ve) m/z 180 (M⁺).

Example 41: 4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-oneExample 41a: 6-allyl-2,2,5,5-tetramethylcyclohexanone

To a solution of the product of Example 3c (500 mg, 3.24 mmol) intetrahydrofuran (6 mL) at −78° C. under argon, was added 2.0 M lithiumdiisopropylamide (1.8 ml, 3.6 mmol) and then the reaction was warmed to−20° C. and stirred for 2 hours. After recooling to −78° C.,hexamethylphosphoramide (0.72 ml, 3.4 mmol) and 3-bromoprop-1-ene (0.6ml, 6.48 mmol) were added and the mixture was stirred for 3 hours. Themixture was diluted with ethyl acetate (60 mL) and the organic layer waswashed with brine (50 mL), dried over anhydrous anhydrous sodium sulfateand concentrated in vacuo. The residue was purified by silica gel columnchromatography to afford the title compound (180 mg, Yield: 29%). ¹H NMR(400 MHz, CDCl₃) δ 5.80-5.76 (m, 1H), 5.00-4.89 (m, 2H), 2.55-2.47 (m,2H), 1.99-1.86 (m, 2H), 1.69-1.59 (m, 2H), 1.38 (m, 1H), 1.18 (s, 3H),1.10 (s, 3H), 1.03 (s, 3H), 0.69 (s, 3H).

Example 41b: 2,2,5,5-tetramethyl-6-(2-oxopropyl)cyclohexanone

A mixture of the product of Example 41a (180 mg, 0.93 mmol) palladiumdichloride (10 mg, 0.05 mmol), cupric acetate hydrate (47 mg, 0.23 mmol)in dimethylacetamide (6 mL) and water (0.9 mL) under oxygen was stirredat room temperature for 3 days. The reaction mixture was concentrated invacuo and the residue purified by silica gel column chromatography toafford the title compound as a yellow oil (50 mg, Yield: 26%). ¹H NMR(400 MHz, CDCl3) δ 3.20 (d, J=10.8 Hz, 1H), 2.89 (dd, J=10.8, 16.8 Hz,1H), 2.24 (s, 3H), 2.13 (dd, J=6.8, 16.8 Hz, 1H), 1.99-1.93 (m, 1H),1.67-1.64 (m, 2H), 1.40 (d, J=13.6 Hz, 1H), 1.24 (s, 3H), 1.03 (s, 3H),1.01 (s, 3H), 0.69 (s, 3H).

Example 41: 4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of the product of Example 41b (50 mg, 0.24 mmol) in xylene(10 mL) under argon, was added potassium hydroxide (6 mg, 0.108 mmol)and the reaction was stirred at reflux for 2 hours. The mixture wasconcentrated under reduced pressure and the residue was purified byprep-TLC to afford the title compound as a yellow solid (30 mg, Yield:65%). Mp=33° C.; R_(f)=0.6 (10:1 petroleum ether/ethyl acetate; ¹H NMR(400 MHz, CDCl₃+D₂O) δ 5.87 (s, 1H), 2.76 (d, J=6.4 Hz, 1H), 2.30-2.18(m, 2H), 1.59 to 1.39 (m, 4H), 1.21 (s, 3H), 1.18 (s, 3H), 1.01 (s, 3H),0.64 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 193 (M+H⁺).

Example 42:(±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxamideExample 42a:(±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxylicacid

To a solution of the product of Example 8 (50 mg, 0.22 mmol) inmethanol/water (5 mL/1 mL) was added sodium hydroxide (44 mg, 1.1 mmol).The mixture was stirred at room temperature overnight and then refluxedfor 1 hour. The reaction mixture acidified to pH=2-3 with 1Nhydrochloric acid, concentrated under reduced pressure and the aqueousresidue extracted with ethyl acetate (60 mL). The organic layer waswashed by brine (30 mL×3), dried over anhydrous anhydrous sodium sulfateand concentrated under reduced pressure to afford the title compound asa white solid (44 mg, Yield: 95%). ¹HNMR (400 MHz, CDCl₃) δ 6.70 (s,1H), 2.52-2.41 (m, 2H), 1.75-1.69 (m, 1H), 1.52-1.41 (m, 3H), 1.34-1.26(m, 5H), 1.16-1.13 (m, 1H), 1.02 (s, 3H), 0.91 (s, 3H).

Example 42:(±)-(3aS,7aS)-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-indene-2-carboxamide

To a solution of the product of Example 42a (41 mg, 0.20 mmol) intetrahydrofuran (5 mL) at 0° C. was added triethylamine (30 μL, 0.22mmol) and isobutyl chloroformate (29 μL, 0.22 mmol) and the reaction wasstirred for 1 hour. Then 25% ammonium hydroxide (40.9 mg, 0.60 mmol) wasadded and the mixture was stirred for 35 minutes. The organics wereextracted with ethyl acetate (60 mL), the organic layer was washed by 2NHCl (30 mL×2), water (30 mL×2) and brine (30 mL×2), dried over anhydroussodium sulfate and concentrated under reduced pressure. Purification ofthe residue by silica column chromatography gave the title compound as awhite solid (30 mg, Yield: 72%). Mp=109.9-112.6° C.; R_(f)=0.3 (1:1petroleum ether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ 6.33 (s, 1H),5.46 (br, s, 2H), 2.45 (d, J=9.6 Hz, 1H), 1.73 (t, J=9.8 Hz, 1H),1.52-1.44 (m, 3H), 1.34-1.25 (m, 5H), 1.18-1.11 (m, 1H), 1.03 (s, 3H),0.91 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 208 (M+H⁺).

Example 43:(±)-(3aR,7aS)-3-tert-butyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 43a:3-hydroxy-4,4-dimethyl-1-(2,6,6-trimethylcyclohex-1-enyl)pentan-1-one

To a solution of the product of Example 28b (324 mg, 1.98 mmol) in drytetrahydrofuran (13 mL) under argon at −78° C. was added drop wiselithium diisopropylamide (1.19 mL, 2.38 mmol). The mixture was stirredfor 1 h, then warmed to 0° C. and stirred for 30 minutes. The reactionsolution was recooled to −78° C. and pivalaldehyde (341.1 mg, 3.96 mmol)was slowly added. The reaction was stirred for 2.5 hours. The mixturewas quenched by the addition of saturated aqueous ammonium chloride (20mL) and after warming to room temperature the mixture was extracted withethyl acetate. The organic phase was dried over anhydrous sodium sulfateand then the organic phase was concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=80/1->40/1) to afford the title compoundas a colorless oil (318 mg, Yield 63%). R_(f)=0.4 (10:1 petroleumether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ 3.78-3.75 (m, 1H), 3.34(s, 1H), 2.76 (d, J=18.0 Hz, 1H), 2.50 (dd, J=18.4, 10.0 Hz, 1H), 1.95(t, J=6.0 Hz, 2H), 1.68-1.62 (m, 2H), 1.57 (s, 3H), 1.45-1.42 (m, 2H),1.06 (d, J=5.2 Hz, 6H), 0.91 (s, 9H).

Example 43b:(E)-4,4-dimethyl-1-(2,6,6-trimethylcyclohex-1-enyl)pent-2-en-1-one

To a stirred solution of the product of Example 43a (175.3 mg, 0.69mmol) in toluene (3.5 mL) was added p-toluenesulfonic acid (6.6 mg, 0.03mmol). The mixture was warmed and stirred at 90° C. for 30 minutes andthen after cooling to room temperature aqueous 0.1N sodium hydroxide (10mL) and water (5 mL) were added. The organics were then extracted withethyl acetate and the organic phase was dried over anhydrous sodiumsulfate. The reaction mixture was concentrated in vacuo and the residuewas purified by silica gel column chromatography (eluent: petroleumether/ethyl acetate=60/1) to afford the title compound as a colorlessoil (132 mg, Yield: 82%). R_(f)=0.45 (10:1 petroleum ether/ethylacetate; ¹H NMR (400 MHz, CDCl₃) δ 6.65 (d, J=16.0 Hz, 1H), 6.11 (d,J=16.0 Hz, 1H), 2.01-1.98 (m, 2H), 1.72-1.66 (m, 2H), 1.48-1.45 (m, 5H),1.07 (s, 9H), 1.01 (s, 6H).

Example 43:(±)-(3aR,7aS)-3-tert-butyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

The product of Example 43b (80 mg, 0.34 mmol) was dissolved in 85%phosphoric acid (0.5 mL) and the mixture was stirred at 90° C. for 2hours. The reaction was cooled to room temperature and water (10 mL) wasadded and then the organics were extracted with ethyl acetate. Theorganic phase was dried over anhydrous sodium sulfate and thenconcentrated under reduced pressure. The residue was purified byprep-TLC to afford the title compound as a colorless oil (10 mg, Yield:12%). R_(f)=0.4 (10:1 petroleum ether/ethyl acetate; ¹H NMR (400 MHz,CDCl₃) δ 5.83 (s, 1H), 2.20-2.14 (m, 1H), 1.86 (s, 1H), 1.66-1.62 (m,1H), 1.60-1.53 (m, 2H), 1.40-1.31 (m, 1H), 1.30 (s, 3H), 1.28 (s, 9H),1.30-1.25 (m, 1H), 1.45 (s, 3H); 0.80 (s, 3H) ppm; Mass spectrum(ESI+ve) m/z 235 (M+H⁺).

Example 44:(±)-(3aR,7aS)-3-cyclopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 44a:(E)-3-cyclopropyl-1-(2,6,6-trimethytcyclohex-1-enyl)prop-2-en-1-one

To a solution of the product of Example 28b (310 mg, 1.89 mmol) in drytetrahydrofuran (13 mL) under argon at −78° C. was drop wise addedlithium diisopropylamide (1.13 mL, 2 mmol). The mixture was stirred for1 hour, then warmed to 0° C. and stirred for 30 minutes. After recoolingto −78° C., cyclopropanecarbaldehyde (264.6 mg, 3.78 mmol) was added tothe reaction mixture. The reaction was stirred for 2.5 hours and thenwas quenched by the addition of saturated aqueous ammonium chloride (20mL). After warming to room temperature, the reaction was extracted withethyl acetate and the organic phase was dried over anhydrous sodiumsulfate and then concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (eluent: petroleumether/ethyl acetate=80/1->40/1) to afford the title compound as a whitesolid (232 mg, Yield: 56%). R_(f)=0.45 (10:1 petroleum ether/ethylacetate; ¹H NMR (400 MHz, CDCl₃) δ 6.18 (s, 1H), 6.17 (d, J=1.6 Hz, 1H),1.96-1.93 (m, 2H), 1.67-1.58 (m, 3H), 1.43-1.39 (m, 5H), 0.99-0.94 (m,8H), 0.68-0.65 (m, 2H).

Example 44:(±)-(3aR,7aS)-3-cyclopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

The product of Example 44a (100 mg, 0.46 mmol) was dissolved in 85%phosphoric acid (0.6 mL) and the mixture was stirred at 60° C. for 3hours. The reaction was cooled to room temperature and water (10 mL) wasadded. The organics were extracted with ethyl acetate and the organicphase was dried over anhydrous sodium sulfate and then concentratedunder reduced pressure. The residue was purified by prep-TLC to affordthe get the title compound as colorless oil (42 mg, Yield: 42%).R_(f)=0.4 (10:1 petroleum ether/ethyl acetate; ¹H NMR (400 MHz, CDCl₃) δ5.38 (s, 1H), 1.86 (s, 1H), 1.81-1.76 (m, 1H), 1.71-1.45 (m, 4H),1.38-1.34 (m, 2H), 1.27 (s, 3H), 1.19 (s, 3H), 1.11-1.03 (m, 2H), 0.88(s, 3H), 0.73-0.69 (m, 2H) ppm; Mass spectrum (ESI+ve) m/z 219 (M+H⁺).

Example 45:(±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneoxime

To a stirred solution of the product of Example 29 (45 mg, 0.19 mmol) inpryidine (4 mL) was added hydroxylamine hydrochloride (40 mg, 0.57 tommol) and the mixture was refluxed overnight. The mixture wasconcentrated under reduced pressure and 1N hydrochloric acid (10 mL) andwater (20 mL) was added. The mixture was extracted with ethyl acetate(30 mL×3). The combined organic phase was washed with 1N hydrochloricacid (20 mL×2) and brine (30 mL×2), dried over anhydrous sodium sulfateand concentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=100/1->50/1) and then by prep-TLC (eluent: petroleum ether/ethylacetate=5/1) gave title compound as a white solid (28 mg, Yield: 62%).Mp=136-138° C.; R_(f)=0.3 (10:1 petroleum ether/ethyl acetate); ¹H NMR(400 MHz, CDCl₃) δ 6.39 (s, 1H), 2.48-2.41 (m, 1H), 2.12 (s, 1H),1.97-1.92 (m, 1H), 1.54-1.50 (m, 1H), 1.41-1.21 (m, 5H), 1.15 (d, J=6.8Hz, 6H), 1.08 (s, 3H), 1.07 (s, 3H), 0.77 (s, 3H) ppm; Mass spectrum(ESI+ve) m/z 236 (M+H⁺).

Example 46:(±)-(3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-one

To a solution of the product of Example 29 (230 mg, 1.11 mmol) inmethanol (3 mL) was added 5% Pd/C (36 mg). The mixture was stirred atroom temperature under an atmosphere of hydrogen overnight. The mixturewas filtered and the filtrate concentrated under reduced pressure.Purification of the residue by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=100/1) gave the title compound as a whitesolid (208 mg, Yield: 84%). Mp=36-38° C.; R_(f)=0.8 (10:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.32-2.24 (m, 1H), 2.02(dd, J=18.8, 12.4 Hz, 1H), 1.87-1.82 (m, 1H), 1.65 (s, 1H), 1.65-1.42(m, 4H), 1.33 (s, 3H), 1.25 (s, 3H), 1.25-1.11 (m, 3H), 1.06 (s, 3H),1.02 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 218.2, 66.2, 50.9, 43.6, 40.4, 36.1, 33.1, 30.2, 29.6, 28.2, 27.1,27.0, 24.8, 21.4, 18.2 ppm; Mass spectrum (ESI+ve) m/z 223 (M+H⁺).

Example 47:(±)-(1S,3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol

A stirred solution of the product of Example 29 (77 mg, 0.35 mmol) indry tetrahydrofuran (4 mL) cooled to −78° C. was added lithium aluminumhydride (80 mg, 2.10 mmol). The reaction was stirred at −78° C. for 2hours. Water (0.2 mL), 15% aqueous sodium hydroxide (0.2 mL) and water(0.6 mL) was added to quench the reaction. Water (20 mL) was added andthe reaction and the mixture was extracted with ethyl acetate (30 mL×3).The combined organic phase was washed with brine (30 mL×2), dried overanhydrous sodium sulfate and then concentrated under reduced pressure.Purification of the residue by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=250/1) gave a white solid (62 mg) that wasfurther purified by prep-HPLC to afford the title compound as a whitesolid (30 mg, Yield: 38%). Mp=95-96° C.; R_(f)=0.5 (10:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 5.32 (s, 1H), 4.58 (t,J=7.2 Hz, 1H), 2.26-2.22 (m, 1H), 1.65-1.61 (m, 1H), 1.53-1.46 (m, 1H),1.39-1.23 (m, 5H), 1.21 (s, 3H), 1.09-1.03 (m, 12H), 0.94 (dt, J=13.2,4.0 Hz, 1H) ppm; Mass spectrum (ESI+ve) m/z 205 (M−H₂O+H⁺).

Example 48:(±)-(3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-oneO-methyl oxime

A solution of the product of Example 46 (22 mg, 0.10 mmol) in pyridine(2 ml) was added O-methylhydroxylamine (50 mg, 0.60 mmol). The reactionwas stirred at 150° C. under microwave irradiation for 1 hour. Themixture was concentrated under reduced pressure, water (20 mL) was addedand the mixture was extracted with ethyl acetate (30 mL×3). The combinedorganic phase was washed with 1N hydrochloric acid (20 mL×2) and brine(30 mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (eluent: petroleum ether) to afford the title compound asa colorless oil (6.6 mg, Yield: 26%). R_(f)=0.5 (100:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 3.84 (s, 3H), 2.64 (dd,J=18.0, 8.0 Hz, 1H), 2.12-2.04 (m, 1H), 2.02 (s, 1H), 1.75-1.72 (m, 1H),1.60-1.52 (m, 2H), 1.44-1.38 (m, 1H), 1.32-1.16 (m, 10H), 1.09 (s, 3H),0.97 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 165.8, 61.4, 60.3, 53.3, 44.3, 34.5, 33.6, 30.9, 30.3, 29.3, 27.2,27.0, 26.9, 24.6, 21.4, 18.0 ppm; Mass spectrum (ESI+ve) m/z 252 (M+H⁺).

Example 49:(±)-(3aS,7aS)-3a,7,7-trimethyloctahydro-1H-indene-2-carboxamide

To a solution of the product of Example 42 (18.5 mg, 0.09 mmol) inmethanol (4 mL) was added 5% Pd/C (20 mg) and then the mixture wasstirred at room temperature under an atmosphere of hydrogen overnight.The mixture was filtered and the filtrate was concentrated under reducedpressure.

Purification of the residue by silica gel column chromatography affordedthe title compound as a white solid (15.5 mg, Yield: 82%). Mp=97.7-99.6°C.; R_(f)=0.35 (1:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 5.31 (bs, 2H), 2.68-2.63 (m, 1H), 2.04-1.91 (m, 2H), 1.77 (d,J=8.8 Hz, 2H), 1.52-1.37 (m, 5H), 1.21-1.12 (m, 2H), 1.12 (s, 3H), 1.03(s, 3H), 0.85 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 178.7, 55.7, 46.2,40.8, 40.4, 34.2, 34.1, 33.0, 31.8, 31.6, 28.8, 27.9, 19.2 ppm; Massspectrum (ESI+ve) m/z 210 (M+H⁺).

Example 50:(±)-(1R,3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-ol

To a stirred solution of the product of Example 46 (60 mg, 0.27 mmol) indry tetrahydrofuran (4 mL) cooled to −78° C. was added lithium aluminumhydride (62 mg, 1.62 mmol). The reaction was stirred at −78° C. for 3hours and then allowed to warm to room temperature and stirred for anadditional 1.5 hours. Water (0.2 mL), 15% aqueous sodium hydroxide (0.2mL) and water (0.6 mL) was added to quench the reaction. Additionalwater (20 mL) was added to the reaction mixture and then the organicswere extracted with ethyl acetate (30 mL×3). The combined organic phasewas washed with brine (30 mL×2), dried over anhydrous sodium sulfate andthen concentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=100/1) gave the title compound as a colorless liquid (45 mg,Yield: 74%). R_(f)=0.8 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 4.31-4.29 (m, 1H), 2.29-2.23 (m, 1H), 1.80-1.54 (m, 5H),1.38-1.25 (m, 3H), 1.19-1.07 (m, 3H), 1.14 (s, 3H), 1.13 (s, 3H), 1.07(s, 3H), 0.97 (d, J=6.4 Hz, 3H), 0.88 (d, J=6.4 Hz, 3H); ¹³C NMR (100MHz, CDCl₃) δ 75.1, 61.2, 56.1, 43.6, 38.5, 36.7, 32.7, 31.0, 30.9,28.7, 28.6, 27.8, 24.4, 21.8, 19.1 ppm; Mass spectrum (El+ve) m/z 224(M⁺).

Example 51:(±)-(3aR,7aS)-3-isopropyl-3a,7,7-trimethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneO-methyl oxime

A solution of the product of Example 29 (70 mg, 0.32 mmol) in pyridine(2 mL) was added O-methylhydroxylamine hydrochloride (160 mg, 1.91mmol). The reaction was stirred at 150° C. under microwave irradiationfor 1 hour. The mixture was concentrated under reduced pressure andwater (20 mL) was added. The reaction mixture was extracted with ethylacetate (30 mL×3) and the combined organic phase was washed with 1Nhydrochloric acid (20 mL×2) and brine (30 mL×2), dried over anhydroussodium sulfate and then concentrated under reduced pressure.Purification of the residue by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=100/1) afforded a 35 mg of a colorlessliquid which was further purified by prep-TLC (eluent: petroleumether/ethyl acetate=100/1) to afford the title compound as a pale yellowliquid (23 mg, Yield: 29%). R_(f)=0.9 (100:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) (major isomer) δ 6.27 (s, 1H), 3.88(s, 3H), 2.45-2.41 (m, 1H), 2.11 (s, 1H), 1.98-1.94 (m, 1H), 1.52-1.21(m, 5H), 1.15-1.07 (m, 12H), 0.71 (s, 3H) ppm; Mass spectrum (ESI+ve)m/z 250 (M+Fr).

Example 52:(±)-(3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-oneoxime

To a solution of the product of Example 46 (39 mg, 0.18 mmol) inpyridine (2 mL) was added hydroxylamine hydrochloride (38 mg, 0.54mmol). The reaction was stirred at 150° C. under microwave irradiationfor 1 hour. The mixture was concentrated under reduced pressure andwater (20 mL) was added. The mixture was extracted with ethyl acetate(30 mL×3) and the combined organic phase was washed with 1N hydrochloricacid (20 mL×2) and brine (20 mL×2), dried over anhydrous sodium sulfateand concentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethyl acetate100:1->20/1) to afford 18 mg of material was purified by crystallizationfrom cold petroleum ether to afford the title compound as a white solid(7 mg, Yield: 16%). Mp=152-153° C.; R_(f)=0.6 (10:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃+D₂O) δ 2.74 (dd, J=18.0,8.0 Hz, 1H), 2.15 (dd, J=18.0, 12.4 Hz, 1H), 2.04 (s, 1H), 1.79-1.74 (m,1H), 1.60-1.51 (m, 3H), 1.37-1.14 (m, 4H), 1.24 (s, 3H), 1.18 (s, 3H),1.09 (s, 3H), 0.99 (d, J=6.8 Hz, 3H), 0.88 (d, J=6.8 Hz, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 167.5, 60.3, 53.2, 44.5, 34.7, 33.6, 30.9, 30.4,28.8, 27.2, 27.1, 27.0, 24.6, 21.4, 18.0 ppm; Mass spectrum (ESI+ve) m/z238 (M+H⁺).

Example 53: 4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of the product of Example 27f (116 mg, 0.64 mmol) intetrahydrofuran (5 mL) at 0° C. was added potassium t-butoxide (71.8 mg,0.64 mmol) and the mixture was stirred for 30 minutes. The reaction wasquenched with saturated aqueous ammonium chloride (3 mL) and the mixturewas extracted with ethyl acetate (100 mL). The organic phase was washedwith water (50 mL×2) and brine (50 mL×2), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. Purification of theresidue by prep-TLC gave the title compound as light yellow oil (8 mg,Yield: 8%). R_(f)=0.3 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 5.85 (s, 1H), 2.91-2.88 (m, 1H), 2.59 (dd, J=18.8, 6.8 Hz,1H), 2.19-2.15 (m, 1H), 1.99 (dd, J=18.8, 2.0 Hz, 1H), 1.75-1.63 (m,3H), 1.41-1.36 (m, 1H), 1.24 (s, 3H), 1.21 (s, 3H), 1.12-1.05 (m, 1H)ppm; Mass spectrum (ESI+ve) m/z 165 (M+H⁺).

Example 54: 4,4-dimethylhexahydro-1H-inden-2(3H)-one

To a solution of a mixture of the product of Example 53 and the productof Example 27 (56 mg, 0.34 mmol) in methanol (5 mL) was added Pd/C (50mg). The reaction mixture was stirred at room temperature overnightunder an atmosphere of hydrogen. The reaction was filtered and thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (eluent: petroleum ether/ethyl acetate=100/1)to afford the title compound as a light yellow oil (27 mg, Yield: 48%).R_(f)=0.6 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃):δ 2.39-2.27 (m, 2H), 2.14-1.99 (m, 4H), 1.58-1.47 (m, 3H), 1.32-1.21 (m,2H), 1.05 (s, 3H), 0.98-0.89 (m, 1H), 0.87 (s, 3H); ¹³C NMR (100 MHz,CDCl₃): 220.02, 47.70, 46.68, 37.63, 33.28, 32.95, 31.32, 30.10, 27.71,26.94, 20.99 ppm; Mass spectrum (ESI+ve) m/z 167 (M+H⁺).

Example 55: 4,4-dimethylhexahydro-1H-inden-2(3H)-one oxime

To a solution of the product of Example 54 (45 mg, 0.27 mmol) in ethanol(5 mL) was added hydroxylamine hydrochloride (37.5 mg, 0.54 mmol) andpyridine (42.7 mg, 0.54 mmol). The mixture was heated to reflux for 2hours. The mixture was concentrated under reduced pressure and water (20ml) was added to the residue. The organics were extracted with ethylacetate (50 mL) and the organic layer was washed with 1N hydrochloricacid (30 mL×2) and brine (30 mL×2), dried over anhydrous sodium sulfateand concentrated under reduced pressure. Purification of the residue bysilica gel column chromatography gave the title compound as a whitesolid (38 mg, Yield: 78%). Mp=67.4-69.6° C.; R_(f)=0.3 (5:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.54-2.39 (m, 2H),2.35-2.14 (m, 3H), 1.79-1.76 (m, 1H), 1.48-1.45 (m, 3H), 1.34-1.26 (m,2H), 1.20-1.17 (m, 1H), 1.01 (s, 3H), 0.96-0.85 (m, 4H); ¹³C NMR (100MHz, CDCl₃) δ 166.6, 166.4, 48.2, 48.0, 38.4, 35.2, 34.8, 34.3, 33.3,33.1, 31.4, 31.3, 30.0, 29.9, 29.8, 27.9, 27.6, 26.9, 26.8, 21.3, 21.1ppm; Mass spectrum (ESI+ve) m/z 182 (M+H⁺).

Example 56: 4,4-dimethyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one Example56a: 3,3-dimethylcyclohex-1-enecarbaldehyde

To a stirred solution of a mixture of the product of Example 27c (2.10g, 10.0 mmol) in dry diethyl ether (50 mL) at −20° C. was added lithiumaluminum hydride (0.57 g, 15.0 mmol) portion wise. The resulting mixturewas stirred for 1 hour during which time the reaction temperature wasallowed to warm to room temperature. The reaction was diluted with ethylacetate (5 mL) and then saturated aqueous ammonium chloride (5 mL) wasadded and the mixture was further diluted with diethyl ether (300 mL).The organic phase was dried over anhydrous magnesium sulfate, filteredand then concentrated under reduced pressure to give a colorless liquid.The material was dissolved in acetone (20 mL) and 2N HCl (0.05 mL) wasadded and the solution was shaken for 1 minute. Solid sodium bicarbonate(50 mg) was added and the solution was concentrated under reducedpressure. The residue was purified by silica gel column chromatographygive the title compound as a colorless liquid (1.15 g, Yield: 83%). ¹HNMR (400 MHz, CDCl₃) δ 9.39 (s, 1H), 6.47 (s, 1H), 2.14 (t, J=6.0 Hz,2H), 1.69-1.63 (m, 2H), 1.53-1.50 (m, 2H), 1.10 (s, 6H).

Example 56b: 1-(3,3-dimethylcyclohex-1-enyl)prop-2-en-1-ol

To a stirred solution of the product of Example 56a (200 mg, 1.45 mmol)in tetrahydrofuran (5 mL) at 0° C. was added vinylmagnesium bromide (3.1ml, 2.17 mmol) and then the mixture was warmed to room temperature andstirred for 1 h. The reaction was quenched with saturated aqueousammonium chloride (10 mL) and the organic were then extracted with ethylacetate (10 mL×3). The combined organic phase was washed brine, driedover anhydrous sodium sulfate and concentrated under reduced pressure toafford the title compound as a colorless oil (256 mg, Yield, 100%). ¹HNMR (400 MHz, CDCl₃) δ 5.90-5.81 (m, 1H), 5.45 (s, 1H), 5.26 (dt,J=17.2, 1.2 Hz, 1H), 5.14 (dt, J=14.4, 1.2 Hz, 1H), 4.45 (d, J=6.0 Hz,1H), 1.95-1.88 (m, 2H), 1.66-1.58 (m, 3H), 1.43-1.39 (m, 2H), 0.98 (s,3H), 0.97 (s, 3H).

Example 56c: 1-(3,3-dimethylcyclohex-1-enyl)prop-2-en-1-one

To a solution of compound of the product of Example 56b (246 mg, 1.47mmol) in dichloromethane (5 mL) at 0° C. was portion wise addedDess-Martin periodinane (1.13 g, 2.66 mmol) and then the mixture waswarmed to room temperature and stirred for 1 hour. Then dichlormethane(20 mL) was added and the mixture was washed with saturated aqueoussodium bicarbonate and brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified byprep-TLC to afford the title compound as a colorless oil (61 mg, Yield:25%). ¹H NMR (400 MHz, CDCl₃) δ 6.94 (dd, J=17.2, 10.8 Hz, 1H), 6.59 (s,1H), 6.24 (dd, J=17.2, 1.8 Hz, 1H), 5.70 (dd, J=10.4, 2.0 Hz, 1H),2.26-2.23 (m, 2H), 1.68-1.65 (m, 2H), 1.48-1.45 (m, 2H), 1.08 (s, 6H).

Example 56: 4,4-dimethyl-2,3,4,5,6,7-hexahydro-1H-inden-1-one

A mixture of the product of Example 56c (40.0 mg, 0.244 mmol) inphosphoric acid (86 mg) and formic acid (0.26 g) was stirred at 80° C.for 2 hours. Dichloromethane (10 mL) was added and the mixture waswashed with saturated aqueous sodium bicarbonate (5 mL×2) and brine (10mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by prep-TLC to afford the titlecompound as a yellow oil (14 mg, Yield: 35%). R_(t)=0.4 (10:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.52-2.50 (m, 2H),2.38-2.35 (m, 2H), 2.11-2.08 (m, 2H), 1.69-1.64 (m, 2H), 1.56-1.53 (m,2H), 1.14 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 209.8, 179.8, 137.3, 38.3,34.5, 27.3, 25.0, 20.6, 18.7 ppm; Mass spectrum (ESI+ve) m/z 165 (M+H⁺).

Example 57:(±)-(3aS,7aS)-2-(methoxymethyl)-3a,7,7-trimethyloctahydro-1H-indene

To a stirred solution of the product of Example 26 (43 mg, 0.22 mmol) indichloromethane (6 mL) at 0° C. was added1,8-bis(dimethylamino)naphthalene (471.5 mg, 2.2 mmol) andtrimethyloxonium tetrafluoroborate (325 mg, 2.2 mmol). The mixture waswarmed to room temperature stirred overnight. The reaction was quenchedwith saturated aqueous sodium bicarbonate (15 mL) and the resultingmixture was extracted with dichloromethane (50 mL). The organic layerwas washed 5% hydrochloric acid (30 mL×3) and brine (30 mL×2), driedover anhydrous sodium sulfate and concentrated in vacuo. Purification ofthe residue by silica gel column chromatography afforded the titlecompound as a colorless oil (10 mg, Yield: 22%). R_(f)=0.6 (50:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 3.27 (s, 3H),3.21 (d, J=7.2 Hz, 2H), 2.15-2.08 (m, 1H), 1.75-1.73 (m, 1H), 1.60 (t,J=12.2 Hz, 1H), 1.51-1.45 (m, 1H), 1.31-1.19 (m, 6H), 1.14-1.10 (m, 2H),1.03 (s, 3H), 0.95 (s, 3H), 0.77 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ78.2, 57.7, 54.6, 45.5, 39.1, 34.7, 34.3, 33.7, 32.4, 30.94, 30.86,28.1, 27.5, 18.6 ppm; Mass spectrum (ESI+ve) m/z 209 (M+H⁺).

Example 58:(±)-(1S,3S,3aS,7aS)-3-isopropyl-3a,7,7-trimethyloctahydro-1H-inden-1-ol

A mixture of the product of Example 47 (30.0 mg, 0.135 mmol) and 10%Pd/C (50 mg) in methanol (7 mL) was stirred at room temperature under ahydrogen atmosphere overnight. The mixture was filtered and concentratedunder reduced pressure. The residue was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=50/1) to affordthe title compound as a colorless oil (20 mg, Yield: 66%). R_(f)=0.5(10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ4.13-4.11 (m, 1H), 1.93-1.85 (m, 1H), 1.66-1.61 (m, 3H), 1.59-1.48 (m,1H), 1.45-1.40 (m, 2H), 1.25-1.20 (m, 5H), 1.17 (s, 3H), 1.08 (s, 3H),1.00 (s, 3H), 0.96 (d, J=6.4 Hz, 3H), 0.83 (d, J=6.4 Hz, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 73.0, 66.7, 55.5, 44.9, 36.2, 35.9, 31.8, 31.6, 29.2,28.9, 27.2, 26.6, 24.5, 21.4, 18.6 ppm; Mass spectrum (ESI+ve) m/z 224(M+H⁺).

Example 59: (±)-(3a5,7aS)-3a,7,7-trimethyloctahydro-1H-inden-1-one

To a solution of the product of Example 35 (50 mg, 0.28 mmol) inmethanol (5 mL) was added Pd/C (30 mg). The solution was stirred at roomtemperature under an atmosphere of hydrogen overnight. The mixture wasfiltered and concentrated under reduced pressure. Purification of theresidue by silica gel column chromatography (eluent: petroleumether/dichloromethane=2/1) afforded the title compound as a colorlesssemisolid (32 mg, Yield: 63%). R_(f)=0.5 (2:1 petroleumether/dichloromethane); ¹H NMR (400 MHz, CDCl₃) δ 2.34-2.27 (m, 1H),2.23-2.16 (m, 1H), 2.05-1.97 (m, 1H), 1.61-1.43 (m, 5H), 1.40-1.31 (m,2H), 1.19-1.12 (m, 1H), 1.06 (s, 3H), 0.97 (s, 3H), 0.93 (s, 3H); ¹³CNMR (100 MHz, CDCl₃) δ 222.2, 64.0, 39.8, 38.9, 36.9, 35.5, 32.5, 32.3,32.1, 31.5, 23.9, 18.2 ppm; Mass spectrum (ESI+ve) m/z 181 (M+H⁺).

Example 60: (±)-(1S,3a5,7aS)-3a,7,7-trimethyloctahydro-1H-inden-1-ol

To a solution of the product of Example 59 (36 mg, 0.20 mmol) inmethanol (5 mL) was added Pd/C (20 mg). The solution was stirred underan atmosphere of hydrogen at room temperature overnight. The mixture wasfiltered and the filtrate was concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=20/1) to afford the title compound as awhite solid (24 mg, Yield: 67%). Mp=59.2-61.3° C.; R₁=0.2 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 4.24 (t, J=6.6Hz, 1H), 2.16-2.10 (m, 1H), 1.76-1.67 (m, 1H), 1.61-1.31 (m, 5H),1.25-1.15 (m, 5H), 1.13 (s, 3H), 1.05 (s, 3H), 1.01 (s, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 76.2, 64.3, 42.0, 41.0, 35.8, 33.2, 32.0, 31.6, 31.0,29.0, 28.1, 18.9 ppm; Mass spectrum (ESI+ve) m/z 165 (M−H₂O+H⁺).

Example 61:(±)-(1R,7aS)-1-isopropyl-4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of lithium diisopropyl amide (0.35 mL, 0.70 mmol) in drytetrahydrofuran (1 mL) at 0° C. under argon was added the solution ofthe product of Example 53 (57 mg, 0.35 mmol) in dry tetrahydrofuran (2mL). Then the solution was stirred at room temperature for 30 minutesafter which it was cooled to −78° C. and isopropyl iodide (0.35 mL, 3.50mmol) was added. The solution mixture was warmed to room temperature andstirred overnight. The reaction was quenched with water and the organicswere extracted with ethyl acetate (50 mL). The combined organic phasewas washed with brine (30 mL×2), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=50/1->20/1) afforded the title compound as a light yellow oil(19 mg, Yield: 26%). R_(f)=0.5 (20:1 petroleum ether/ethyl acetate); ¹HNMR (400 MHz, CDCl₃) δ 5.80 (s, 1H), 2.63-2.58 (m, 1H), 2.26-2.21 (m,1H), 2.14-2.10 (m, 1H), 1.91-1.89 (m, 1H), 1.75-1.64 (m, 3H), 1.40-1.31(m, 1H), 1.21 (s, 3H), 1.19 (s, 3H), 1.15-1.08 (m, 1H), 1.00 (d, J=6.8Hz, 1H), 0.77 (d, J=6.8 Hz, 1H) ppm; Mass spectrum (ESI+ve) m/z 207(M+H⁺).

Example 62: (±)-(3aS,7aS)-3a,7,7-trimethylhexahydro-1H-inden-2(3H)-oneoxime

To a solution of the product of Example 40 (50 mg, 0.28 mmol) in ethanol(5 mL) was added hydroxylamine hydrochloride (39 mg, 0.56 mmol) andpyridine (44 mg, 0.56 mmol). The mixture was heated to reflux for 2hours. The reaction mixture was concentrated under reduced pressure andthe residue was portioned between water and ethyl acetate (50 mL). Theorganic phase was washed with 1N hydrochloric acid (30 mL×2) and brine(30 mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure. Purification of the residue by silica gel columnchromatography gave the title compound as a white solid (34 mg, Yield:62%). Mp=124.7-126.1° C.; R_(f)=0.3 (5:1 petroleum ether/ethyl acetate);¹H NMR (400 MHz, CDCl₃) (Major isomer) δ 8.32 (br, 1H), 2.60-2.17 (m,4H), 1.59-1.53 (m, 2H), 1.46-1.40 (m, 1H), 1.37-1.30 (m, 1H), 1.28-1.20(m, 3H), 1.18 (s, 3H), 1.05 (s, 3H), 0.87 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) (Isomers) δ 165.0, 164.9, 53.0, 52.6, 47.6, 44.3, 39.7, 39.3,34.2, 33.8, 33.5, 32.9, 32.4, 32.1, 32.0, 30.6, 30.5, 30.0, 28.9, 28.7,27.6, 27.1, 18.9, 18.7 ppm; Mass spectrum (ESI+ve) m/z 196 (M+H⁺).

Example 63: (±)-(3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-2-ol

To a 0° C. stirred solution of the product of Example 40 (100 mg, 0.55mmol) in methanol (1 mL) and tetrahydrofuran (7 mL) was added sodiumborohydride (25 mg, 0.66 mmol) and the reaction was warmed to roomtemperature and stirred for 2 hours. The reaction was quenched with 5%hydrochloric acid (3 mL) and then the organics were extracted with ethylacetate (50 mL). The organic layer was washed with brine (30 mL×2),dried over anhydrous sodium sulfate and concentrated under reducedpressure. Purification of the residue by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=10/1) afforded thetitle compound as a colorless oil (95 mg, Yield: 94%). R_(f)=0.3 (10:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) (Major isomer) δ4.30-4.24 (m, 1H), 2.20-2.13 (m, 1H), 1.83 (dd, J=14.2, 5.0 Hz, 2H),1.72-1.40 (m, 6H), 1.36-1.15 (m, 3H), 1.08 (s, 3H), 1.02 (s, 3H), 0.83(s, 3H); ¹³C NMR (100 MHz, CDCl₃) (Major isomer) δ 70.7, 54.6, 53.3,40.0, 39.4, 34.4, 34.2, 32.0, 31.6, 29.0, 28.0, 19.4; (Minor isomer) δ70.3, 54.2, 52.6, 41.1, 39.1, 34.3, 34.2, 31.7, 31.4, 28.9, 28.0, 19.2ppm; Mass spectrum (ESI+ve) m/z 165 (M−H₂O+H⁺).

Example 64:(i)-(3aS,7aS)-2,3,4,4-tetramethyl-3a,4,5,6,7,7a-hexahydro-H-inden-1-oneExample 64a: 1-(3,3-dimethylcyclohex-1-enyl)-2-methylbut-2-en-1-ol

To a solution of the product of Example 56a (300 mg, 2.17 mmol) intetrahydrofuran (8 mL) at 0° C. was added 1-methyl-1-propenylmagnesiumbromide (0.5 M in tetrahydrofuran, 6.7 mL, 3.26 mmol) and the reactionwas then warmed to room temperature and stirred for 1.5 hours. Themixture was quenched with saturated aqueous ammonium chloride and theorganics were extracted with ethyl acetate. The organic layer was washedwith brine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography to give the title compound as a colorless oil (252 mg,Yield: 60%). R_(f)=0.6 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) (Major isomer) δ 5.50 (s, 1H), 5.42 (q, 1H J=6.8 Hz, 1H),4.92 (d, J=3.2 Hz, 1H), 1.76-1.47 (m, 11H), 1.42-1.39 (m, 2H), 0.99 (s,3H), 0.98 (s, 3H) ppm.

Example 64b: 1-(3,3-dimethylcyclohex-1-enyl)-2-methylbut-2-en-1-one

To a solution of the product of Example 64a (230 mg, 1.18 mmol) indichloromethane (4 mL) at 0° C. was added Dess-Martin periodinane (1.35g, 3.19 mmol) and the reaction mixture was warmed to room temperatureand stirred for 1 hour. The mixture was diluted with petroleum ether andfiltered. The filtrate was concentrated under reduced pressure and theresidue purified by silica gel column chromatography to afford the titlecompound as a colorless oil (207 mg, Yield: 91%). R_(f)=0.5 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) (Major isomer) δ6.52 (s, 1H), 5.61-5.55 (m, 1H), 2.21 (dt, J₁=6.0 Hz, J₂=1.6 Hz, 2H),1.85-1.84 (m, 3H), 1.70-1.64 (m, 2H), 1.52-1.45 (m, 5H), 1.05 (s, 6H)ppm; Mass spectrum (ESI+ve) m/z 193 (M+H⁺).

Example 64:(±)-(3a3,7aS)-2,3,4,4-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

To the product of Example 64b (95 mg, 0.49 mmol) was added phosphoricacid (1.5 mL) and the mixture was stirred at room temperature for 6hours. The mixture was diluted with ethyl acetate and adjusted to pH=8with saturated aqueous sodium carbonate and then the phases wereseparated. The aqueous layer was extracted with ethyl acetate. Thecombined organic phase was washed with brine, dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography to afford the titlecompound as a light yellow oil (40 mg, Yield: 42%). R_(f)=0.3 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.56 (d, J=6.0Hz, 1H), 2.35 (t, J=6.8 Hz, 1H), 2.29-2.25 (m, 1H), 2.07 (d, J=0.4 Hz,3H), 1.71 (s, 3H), 1.44-1.22 (m, 5H), 1.16 (s, 3H), 0.45 (s, 3H) ppm;Mass spectrum (ESI+ve) m/z 193 (M+H⁺).

Examples 65:(±)-(3aR,7aS)-2,3,3a,7,7-pentamethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-oneExample 65a: 2-methyl-1-(2,6,6-trimethylcyclohex-1-enyl)but-2-en-1-ol

To a solution of 2,6,6-trimethylcyclohex-1-enecarbaldehyde (300 mg, 1.97mmol) in tetrahydrofuran (7 mL) at −78° C. was added1-methyl-1-propenylmagnesium bromide (0.5 M in tetrahydrofuran, 5.92 mL,2.96 mmol) and then the mixture was warmed to room temperature andstirred for 2 hours. The mixture was quenched with saturated aqueousammonium chloride and then extracted with ethyl acetate. The combinedorganic phase was washed with brine, dried over anhydrous sodium sulfateand concentrated under reduced pressure. Purification of the residue bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=80/1) to afford the title compound as a colorless oil (256 mg,Yield: 62%). R_(f)=0.35 (20:1 petroleum ether/ethyl acetate); ¹H NMR(400 MHz, CDCl₃) (Major isomer) δ 5.44-5.38 (m, 1H), 5.13 (s, 1H),1.98-1.94 (m, 2H), 1.82 (s, 3H), 1.79 (d, J=8.0 Hz, 3H), 1.69 (s, 3H),1.65-1.54 (m, 2H), 1.45-1.43 (m, 2H), 1.13 (s, 3H), 0.91 (s, 3H) ppm.

Example 65b: 2-methyl-1-(2,6,6-trimethylcyclohex-1-enyl)but-2-en-1-one

To a solution of the product of Example 65a (250 mg, 1.20 mmol) in todichloromethane (8 mL) at 0° C. was added sodium bicarbonate (100.9 mg,1.20 mmol) and Dess-Martin periodinane (1.0 g, 2.40 mmol). The mixturewas warmed to room temperature and stirred for 2 hours. Saturatedaqueous sodium bicarbonate (20 mL) was added to the reaction mixture andthen the organics were extracted with dichloromethane. The organic phasewas washed with brine, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (eluent: petroleum ether/ethyl acetate=90/1)to afford 182 mg of a colorless oil which was further purified byprep-TLC to give the title compound as a mixture of isomers as acolorless oil (165 mg, Yield: 67%). R_(f)=0.5 (20:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 6.03-5.98 (m, 1H),2.00-1.93 (m, 5H), 1.90-1.89 (m, 3H), 1.70-1.68 (m, 2H), 1.56 (s, 3H),1.49-1.45 (m, 2H), 1.07 (s, 6H) ppm.

Examples 65:(±)-(3aR,7aS)-2,3,3a,7,7-pentamethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

The product of Example 65b (60 mg, 0.29 mmol) was dissolved inphosphoric acid (0.5 mL) and the mixture was stirred at room temperaturefor 3 hours. To the solution was added water (3 mL) and the resultingmixture was extracted with ethyl acetate. The combined the organic phasewas dried over anhydrous sodium sulfate and then concentrated underreduced pressure. The residue was purified by prep-TLC to afford thetitle compound as a colorless oil (42 mg, Yield: 70%). R_(f)=0.4 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 1.90 (s, 3H),1.80 (s, 1H), 1.64 (s, 3H), 1.64-1.47 (m, 4H), 1.37-1.34 (m, 2H), 1.21(s, 3H), 1.13 (s, 3H), 0.84 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 207(M+H⁺).

Example 66:(±)-(1R,3aR,7aS)-2,3,3a,7,7-pentamethyl-3a,4,5,6,7,7a-hexahydro 1Hinden-1-ol

To a mixture of lithium aluminum hydride (5.5 mg, 0.15 mmol) in drytetrahydrofuran (1 mL) under argon at 0° C. was added a solution of theproduct of Example 65 (20 mg, 0.097 mmol) in dry tetrahydrofuran. Thereaction was warmed to room temperature stirred for 2 hours. Thereaction was quenched by the addition of the mixture of sodium sulfateand water. The mixture was filtered and then concentrated under reducedpressure. The residue combined with the crude product was purified bysilica gel column chromatography (eluent: petroleum ether/ethylacetate=100/1->80/1) to afford the title compound as a white solid (8.1mg, Yield: 40%). Mp=73-75° C.; R_(f)=0.3 (20:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 4.34-4.31 (m, 1H), 1.62 (s, 3H),1.57-1.46 (m, 4H), 1.34-1.21 (m, 6H), 1.15 (s, 3H), 1.09 (s, 3H), 1.08(s, 3H), 0.85 (dt, J=12.8, 4.0 Hz, 1H) ppm; Mass spectrum (ESI+ve) m/z191 (M−H₂O+H⁺).

Example 67:(±)-(1R,7aS)-1,4,4-trimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a mixture of the product of Example 53 (39 mg, 0.24 mmol) intetrahydrofuran (2 mL) at −78° C. under argon was added lithiumdiisopropylamide (2.0 M, 0.24 ml, 0.48 mmol). The mixture was warmed toroom temperature and stirred for 1 hour and then cooled to −78° C.Methyl iodide (0.15 mL) was added to the mixture and then the reactionwas again stirred at room temperature for 3 hours. The reaction wasquenched with saturated aqueous ammonium chloride and then the mixturewas extracted with ethyl acetate (20 mL×3) and the combined organicphase was washed with brine (20 mL), dried over anhydrous sodium sulfateand concentrated under reduced pressure. The residue was purified byprep-TLC to give the title compound as a yellow oil (14 mg, Yield: 33%).R, =0.8 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl3) δ5.81 (s, 1H), 2.43-2.38 (m, 1H), 2.23-2.19 (m, 1H), 1.97-1.92 (m, 1H),1.71-1.60 (m, 3H), 1.41-1.33 (m, 1H), 1.22 (s, 3H), 1.17-1.16 (m, 6H),1.13-1.02 (m, 1H) ppm; Mass spectrum (ESI+ve) m/z 179 (M+H⁺).

Example 68: (±)-(1R,3aS,7aS)-3a,7,7-trimethyloctahydro-1H-inden-1-ol

To a stirred solution of the product of Example 59 (45 mg, 0.25 mmol) inanhydrous tetrahydrofuran (4 mL) under argon at −78° C. was addedlithium aluminum hydride (47 mg, 1.25 mmol). The reaction was allowed towarm to room temperature and stirred for 2 hours. The mixture wasdiluted with diethyl ether (20 mL) and wet sodium sulfate was added toquench the reaction. The resulting mixture was stirred for another 30minutes and then filtered. The filtrate was concentrated under reducedpressure and the residue was purified by silica gel columnchromatography (eluent: petroleum ether/ethyl acetate=50:1->10:1) toafford the title compound as a colorless liquid. R_(f)=0.6 (20:1petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 4.38 (dd,J=7.6, 4.0 Hz, 1H), 1.83-1.72 (m, 2H), 1.64-1.53 (m, 5H), 1.49-1.43 (m,2H), 1.25-1.21 (m, 1H), 1.16 (s, 3H), 1.14 (d, J=4.0 Hz, 1H), 1.13 (s,3H), 1.09 (d, J=4.0 Hz, 1H), 1.03 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ78.4, 59.2, 41.8, 39.5, 39.2, 37.7, 33.2, 32.1, 31.2, 30.8, 30.4, 19.6ppm; Mass spectrum (ESI+ve) m/z 165 (M−H₂O+H⁺).

Examples 69a and 69b:(+)-4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one (69a) and(−)-4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one (69b)

The product of Example 41 (80 mg, 0.42 mmol) was resolved using chiralcolumn (Chiralpark AY-H 5 cm ID×25 cm Length) HPLC chromatography(eluent: hexane/ethanol=95:5) to afford the title compound (69a) as ayellow solid (32 mg, Yield: 40%). Mp=35-38° C.; ¹H NMR (400 MHz,CDCl₃+D₂O) δ 5.87 (d, J=1.6 Hz, 1H), 2.77 (dt, J=6.8, 1.8 Hz, 1H), 2.32(dd, J=19.0, 6.6 Hz, 1H), 2.16 (dd, J=19.0, 2.2 Hz, 1H), 1.69-1.36 (m,4H), 1.21 (s, 3H), 1.18 (s, 3H), 1.02 (s, 3H), 0.64 (s, 3H) ppm; Massspectrum (ESI+ve) m/z 193 (M+H⁺); [α]_(D) ²⁵=+83.6° (c=0.61,dichloromethane). The title compound (69b) as a yellow solid (19.7 mg,Yield: 25%). [α]_(D) ²⁵=−86.1° (c=0.36, dichloromethane).

Example 70: (3aR,7aS) and(3aS,7aR)-4,4,7,7-tetramethylhexahydro-1H-inden-2(3H)-one

To a solution of the product of Example 41 (110 mg, 0.57 mmol) inmethanol (10 mL) was added 5% Pd/C (20 mg). The mixture was stirred atroom temperature overnight under an atmosphere of hydrogen. The mixturewas filtered and the filtrate concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to afford thetitle compound as a white solid (80 mg, Yield: 72%). R_(f)=0.7 (10:1petroleum ether/ethyl is acetate); ¹H NMR (400 MHz, CDCl₃) δ 2.25-2.09(m, 6H), 1.39-1.32 (m, 4H), 0.97 (s, 6H), 0.83 (s, 6H) ppm; Massspectrum (ESI+ve) m/z 195 (M+H⁺).

Example 71:(±)-(3R,3aR)-3,4,4-trimethyl-2,3,3a,4,5,6-hexahydro-1H-inden-1-oneExample 71a: 1-(3,3-dimethylcyclohex-1-enyl)but-2-yn-1-ol

To a mixture of the product of 56a (700 mg, 5.0 mmol) in tetrahydrofuran(10 mL) at −78° C. under argon was added prop-1-ynylmagnesium bromide(0.5 M, 14.1 ml, 7.05 mmol) dropwise and then the reaction was warmed toroom temperature and stirred for 3 hours. The reaction was quenched withsaturated aqueous ammonium chloride and the organics were extracted withethyl acetate (150 mL). The organic phase was washed with brine (50mL×2), dried over anhydrous sodium sulfate and concentrated underreduced pressure to give the title compound as a colorless oil (844 mg,Yield: 95%). ¹H NMR (400 MHz, CDCl₃) δ 5.56 (s, 1H), 4.68 (d, J=2.4 Hz,1H), 2.12-2.04 (m, 2H), 1.88 (d, J=2.0 Hz, 3H), 1.72-1.64 (m, 3H),1.43-1.40 (m, 2H), 0.99 (s, 3H), 0.98 (s, 3H).

Example 71b: (E)-1-(3,3-dimethylcyclohex-1-enyl)but-2-en-1-ol

To a mixture of the product of Example 71a (844 mg, 4.73 mmol) intetrahydrofuran (25 mL) at 0° C. under argon was added lithium aluminumhydride (720 mg, 19 mmol). The reaction was warmed to room temperatureand stirred overnight. The reaction was quenched by water and then theorganics were extracted with ethyl acetate (20 mL×4). The combinedorganic phase was washed with water (100 mL), brine (100 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography to afford the titlecompound as a as colorless oil (300 mg, Yield: 35%). ¹H NMR (400 MHz,CDCl₃) δ 5.71-5.66 (m, 1H), 5.52-5.46 (m, 1H), 5.43 (s, 1H), 4.38 (d,J=6.8 Hz, 1H), 1.94-1.86 (m, 2H), 1.71 (dd, J=6.4, 0.8 Hz, 3H),1.65-1.61 (m, 2H), 1.48 (s, 1H), 1.42-1.38 (m, 2H), 0.97 (s, 3H), 0.97(s, 3H) ppm.

Example 71c: (E)-1-(3,3-dimethylcyclohex-1-enyl)but-2-en-1-one

To a mixture of the product of Example 71b (300 mg, 1.7 mol) indichloromethane (10 mL) at 0° C. was added manganese dioxide (1.45 g, 17mmol) and then it was warmed to room temperature and stirred for 3 days.The mixture was filtered and the filtrate was concentrated under reducedpressure. The residue was purified by silica gel chromatography to givethe title compound as a colorless oil (121 mg, Yield: 40%). ¹H NMR (400MHz, CDCl₃) δ 6.89-6.84 (m, 1H), 6.68 (dd, J=1.6 Hz 15.2 Hz, 1H), 6.53(s, 1H), 2.25-2.21 (m, 2H), 1.91 (dd, J=1.6 Hz 6.8 Hz, 3H), 1.68-1.62(m, 2H), 1.47-1.44 (m, 2H), 1.07 (s, 6H) ppm.

Example 71:(±)-(3R,3aR)-3,4,4-trimethyl-2,3,3a,4,5,6-hexahydro-1H-inden-1-one

A mixture of the product of Example 71c (120 mg, 0.67 mol) in phosphoricacid (2 mL) was stirred at room temperature for 3 hours. Water was thenadded to the mixture and the organics were extracted with ethyl acetate(20 mL×3). The combined organic phase was washed with water (20 mL),brine (20 ml), dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gelchromatography to afford the title compound as a colorless oil (78 mg,Yield: 65%). R_(f)=0.6 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 6.69 (dd, J=2.8, 6.4 Hz, 1H), 2.42 (dd, J=12.4, 23.2 Hz,1H), 2.24-2.06 (m, 3H), 1.99-1.90 (m, 2H), 1.50-1.34 (m, 2H), 1.25 (d,J=6.0 Hz, 3H), 1.16 (s, 3H), 0.77 (s, 3H) ppm; Mass spectrum (ESI+ve)m/z 179 (M+H⁺).

Example 72:(±)-(1S,3aS,7aS)-2,3,4,4-tetramethyl-3a,4,5,6,7,7a-hexahydro-1H-inden-1-ol

To a solution of the product of Example 64 (100 mg, 0.52 mmol) intetrahydrofuran (2 mL) at 0° C. was added lithium aluminum hydride (118mg, 3.12 mmol) and the reaction was, stirred at 0° C. for 3 hours. Thereaction was quenched with saturated aqueous ammonium chloride andfiltered. The filtrate was extracted with ethyl acetate and the combinedorganic phase was washed with brine, dried over anhydrous sodium sulfateand then concentrated under reduced pressure. The residue was purifiedby silica gel column chromatography to afford the title compound as alight brown oil (20.7 mg, Yield: 20%). R_(f)=0.3 (10:1 petroleumether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 4.29 (s, 1H), 2.15 (d,J=7.6 Hz, 1H), 1.92-1.87 (m, 2H), 1.71 (s, 3H), 1.66 (s, 3H), 1.44-1.17(m, 6H), 1.01 (s, 3H), 0.58 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 136.5,133.9, 80.4, 53.9, 47.3, 39.8, 32.3, 32.1, 23.5, 22.1, 17.1, 15.0, 9.2ppm; Mass spectrum (ESI+ve) m/z 177 (M−H₂O+H⁺).

Example 73: (±)-(3a5,7aS)-2,3,4,4-tetramethyloctahydro-1H-inden-1-oneExample 73a: (±)-(1R,3aS,7aS)-2,3,4,4-tetramethyloctahydro-1H-inden-1-ol

To the product of Example 64 (100 mg, 0.52 mmol) in tetrahydrofuran (2mL) at 0° C. was added lithium aluminum hydride (118 mg, 3.12 mmol) andthe reaction was stirred at 0° C. for 3 hours. The reaction was quenchedwith saturated aqueous ammonium chloride and filtered. The filtrate wasextracted with ethyl acetate and the combined organic phase was washedwith brine, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography on silica to give the title compound as a colorless oil(8.9 mg, Yield: 9%).

Example 73: (±)-(3aS,7aS)-2,3,4,4-tetramethyloctahydro-1H-inden-1-one

To the product of Example 73a (37 mg, 0.19 mmol) in tetrahydrofuran (2mL) at 0° C. was added Dess-Martin periodinane (121 mg, 0.28 mmol) andstirred at 0° C. for 30 minutes. The mixture was added to petroleumether and and filtered. The filtrate was concentrated under reducedpressure and the residue was purified by silica gel columnchromatography to afford the title compound as a colorless oil (8.5 mg,Yield: 23%). R_(f)=0.6 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 2.49-2.36 (m, 3H), 1.96-1.93 (m, 2H), 1.59-1.24 (m, 4H),1.12 (s, 3H), 1.07-1.04 (m, 1H), 1.02-1.00 (m, 6H), 0.97 (d, J=7.2 Hz,3H); ¹³C NMR (100 MHz, CDCl₃) δ 223.3, 51.5, 47.5, 43.2, 37.3, 35.3,31.6, 30.8, 27.9, 25.9, 20.5, 15.1, 9.2 ppm; Mass spectrum (ESI+ve) m/z195 (M+H⁺).

Example 74: (±)-(3S,3aR,7aR)-3,4,4-trimethyloctahydro-1H-inden-1-oneExample 74a: (±)-(3R,3aS,7aS)-3,4,4-trimethyloctahydro-1H-inden-1-ol

The mixture of the product of Example 71 (70 mg, 0.40 mmol) in methanol(4 mL) was added Raney-Ni and the reaction was stirred at roomtemperature over for 2.5 days under an atmosphere of hydrogen. Themixture was filtered and the filtrate was concentrated under reducedpressure. The residue was purified by silica gel chromatography toafford the title compound as a colorless oil (25 mg, Yield: 34%). ¹H NMR(400 MHz, CDCl₃+D₂O) δ 4.20 (dd, J=8.8 Hz, 14.4 Hz, 1H), 2.18-2.08 (m,1H), 2.01-1.90 (m, 2H), 1.61-1.53 (m, 4H), 1.48-1.13 (m, 4H), 1.02 (d,J=6.4 Hz, 3H), 0.95 (s, 3H), 0.88 (s, 3H) ppm.

Example 74: (±)-(3R,3aS,7aR)-3,4,4-trimethyloctahydro-1H-inden-1-one

To the solution of the product of Example 74a (25 mg, 0.14 mmol) indichloromethane (3 mL) at room temperature was added sodium bicarbonate(12 mg, 0.14 mmol) and Dess-Martin periodinane (88 mg, 1.20 mmol) andthe reaction was stirred overnight. The mixture was diluted withdichloromethane and washed with saturated sodium bicarbonate and brine.The organic phase was dried over anhydrous sodium sulfate and thenconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to afford the title compound as a colorlessoil (10 mg, Yield: 39%). R_(f)=0.6 (10:1 petroleum ether/ethyl acetate);¹H NMR (400 MHz, CDCl₃) δ 2.50 (dd, J=9.2 Hz, 19.2 Hz, 1H), 2.42 (dd,J=6.4 Hz, 13.2 Hz, 1H), 2.36-2.30 (m, 1H), 1.87-1.76 (m, 2H), 1.64 (dd,J=4.8 Hz, 8.0 Hz, 1H), 1.49-1.33 (m, 4H), 1.20-1.14 (m, 4H), 0.96 (s,3H), 0.78 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 219.8, 53.0, 47.1, 45.2,38.1, 32.4, 30.3, 28.5, 25.4, 23.3, 22.1, 19.2 ppm; Mass spectrum(ESI+ve) m/z 181 (M+H⁺).

Example 75: 7a-ethyl-4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-oneExample 75a: ethyl 1-ethyl-2-oxocyclohexanecarboxylate

To a stirred suspension of dry potassium carbonate (2.21 g, 16.0 mmol)in anhydrous acetone (45 mL) under argon was added ethyl2-oxocyclohexanecarboxylate (1.36 g, 8.0 mmol) followed by ethyl iodide(2.0 mL, 24.9 mmol). The reaction was heated to reflux for 7 hours afterwhich another portion of ethyl iodide (2.0 mL, 24.9 mmol) was added andthe reaction was refluxed overnight. The cooled reaction mixture wasdiluted with diethyl ether (200 mL) and then filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified bycolumn chromatography (eluent: petroleum ether/ethylacetate=300/1->50/1) to give the title compound as a colorless liquid(870 mg, Yield: 55%). ¹H NMR (400 MHz, CDCl₃) δ 4.21 (q, J=7.2 Hz, 2H),2.54-2.40 (m, 3H), 2.04-1.89 (m, 2H), 1.80-1.57 (m, 4H), 1.45-1.38 (m,1H), 1.26 (t, J=7.2 Hz, 3H), 0.85 (t, J=7.4 Hz, 3H) ppm.

Example 75b: ethyl 1-ethyl-3,3-dimethyl-2-oxocyclohexanecarboxylate

To a solution of potassium tert-butoxide (1.46 g, 13.02 mmol) inanhydrous tetrahydrofuran (15 mL) at −20° C. under argon was addeddropwise a solution of the product of Example 75a (860 mg, 4.34 mmol) inanhydrous tetrahydrofuran (6 mL) over 15 minutes. The resulting mixturewas stirred at −20° C. for 15 minutes after which time methyl iodide(1.7 mL, 27.3 mmol) was added dropwise at −20° C. The mixture was thenallowed to warm gradually to room temperature and stirred for 2 hours.The mixture was poured into cooled half-saturated aqueous ammoniumchloride (100 mL) and then extracted with diethyl ether (80 mL×3). Thecombined organic phase was washed with brine (150 mL), dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (eluent:petroleum ether/ethyl acetate=50/1) afforded the title compound as acolorless oil (768 mg, Yield: 78%). ¹H NMR (400 MHz, CDCl₃) δ 4.22-4.05(m, 2H), 2.60-2.54 (m, 1H), 2.11-1.95 (m, 2H), 1.74-1.68 (m, 1H),1.66-1.51 (m, 3H), 1.31 (dt, J=13.2, 4.4 Hz, 1H), 1.23 (t, J=7.2 Hz,3H), 1.08 (s, 3H), 1.07 (s, 3H), 0.78 (t, J=7.4 Hz, 3H) ppm.

Example 75c: 6-ethyl-2,2-dimethylcyclohexanone

To a solution of the product of Example 75b (165 mg, 0.73 mmol) inmethanol (2.34 mL) in a microwave vessel was added aqueous potassiumhydroxide (6.25 M in water, 0.58 mL, 3.625 mmol). The vessel was sealedand heated to 110° C. for 6 hours. The mixture was diluted with water(25 mL) and extracted with diethyl ether (25 mL×7) and the combinedorganic phase was dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by flash chromatography(eluent: light petroleum ether/ethyl acetate=100/1) to afford the titlecompound as a light yellow oil (100 mg, Yield: 89%). ¹H NMR (400 MHz,CDCl₃) δ 2.47-2.39 (m, 1H), 2.15-2.08 (m, 1H), 1.92-1.73 (m, 3H),1.71-1.63 (m, 1H), 1.59-1.50 (m, 1-H), 1.31-1.22 (m, 2H), 1.18 (s, 3H),1.04 (s, 3H), 0.88 (t, J=7.4 Hz, 3H) ppm.

Example 75d: 2-allyl-2-ethyl-6,6-dimethylcyclohexanone

Toa solution of the product of Example 75c (95 mg, 0.62 mmol) intert-butanol (1 ml) in a microwave vessel was added potassiumtert-butoxide (139 mg, 1.24 mmol) and 3-iodoprop-1-ene (0.17 mL, 1.86mmol). The vessel was sealed and heated to 100° C. overnight. The cooledreaction mixture was transferred to a round bottom flask andconcentrated under reduced pressure. The residue was dissolved in ethylacetate (25 mL), washed with brine (25 mL), dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by flash chromatography (eluent: petroleum ether/ethylacetate=100/1) to afford the title compound as a light yellow oil (71mg, Yield: 59%). ¹H NMR (400 MHz, CDCl₃) δ 5.67-5.56 (m, 1H), 5.04-4.99(m, 2H), 2.34 (dd, J=13.8, 6.6 Hz, 1H), 2.17 (dd, J=13.8, 8.2 Hz, 1H),1.82-1.61 (m, 6H), 1.56-1.49 (m, 2H), 1.10 (s, 3H), 1.08 (s, 3H), 0.75(t, J=7.4 Hz, 3H) ppm.

Example 75e: 2-ethyl-6,6-dimethyl-2-(2-oxopropyl)cyclohexanone

To a solution of the product of Example 75d (70 mg, 0.36 mmol) indimethylacetamide (1.4 mL) and water (0.2 mL) was added palladiumdichloride (6 mg, 0.036 mmol) and cupric acetate hydrate (18 mg, 0.09mmol). The mixture was cooled to −78° C. and then evacuated andback-filled with an atmosphere of oxygen. The mixture was warmed to roomtemperature and stirred vigorously for approximately 4 days. The mixturewas directly subjected to flash column chromatography (eluent: petroleumether/ethyl acetate=50/1->20/1) to afford the title compound as acolorless oil (64 mg, Yield: 84%). ¹H NMR (400 MHz, CDCl₃) δ 3.25 (d,J=18.0 Hz, 1H), 2.24 (d, J=18.4 Hz, 1H), 2.08 (s, 3H), 1.97-1.75 (m,3H), 1.69-1.61 (m, 4H), 1.57-1.45 (m, 1H), 1.17 (s, 3H), 1.10 (s, 3H),0.79 (t, J=7.4 Hz, 3H) ppm.

Example 75: 7a-ethyl-4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a stirred solution of the product of Example 75e (60 mg, 0.29 mmol)in xylenes (2 mL) was added powdered potassium hydroxide (8 mg, 0.14mmol). The reaction was heated to 110° C. for 1 hour. The mixture waspurified by flash column chromatography (eluent: petroleum ether/ethylacetate=50/1->20/1) to give a 55 mg of a colorless oil which was furtherpurified by silica gel column chromatography (eluent: petroleumether/dichloromethane=10/1->1/2) to give 51 mg of a colorless oil whichwas further purified by prep-TLC (eluent: petroleum ether/ethylacetate=10/1) to afford the title compound as a colorless oil (23 mg,Yield: 41%). R_(f)=0.3 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400MHz, CDCl₃) δ 5.93 (s, 1H), 2.34 (d, J=18.0 Hz, 1H), 2.11 (d, J=18.4 Hz,1H), 2.01-1.78 (m, 3H), 1.68-1.53 (m, 3H), 1.45-1.35 (m, 2H), 1.23 (s,3H), 1.20 (s, 3H), 0.74 (t, J=7.4 Hz, 3H) ppm; Mass spectrum (ESI+ve)m/z 193 (M+H⁺).

Example 76:(±)-(1S,7aR)-1-ethyl-4,4-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

To a solution of the product of Example 53 (76 mg, 0.46 mmol) intetrahydrofuran (4 mL) at −78° C. was added lithium diisopropylamide(2.0 M, 0.92 mmol, 0.46 mL) and the mixture was warmed to roomtemperature and stirred for 30 minutes. The reaction mixture wasrecooled to 78° C. and iodoethane (717 mg, 0.37 mL, 4.6 mmol) was added.The mixture was gradually warmed to room temperature and then stirredfor 3.5 hours. The reaction was quenched with saturated aqueous ammoniumchloride (20 mL) and the organics were extracted with ethyl acetate (10mL×3). The combined organic phase was washed with brine (10 mL) and thenconcentrated under reduced pressure. The residue was purified byprep-TLC to afford the title compound as a colorless oil (36 mg, Yield:38%). R_(f)=0.4 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 5.80 (s, 1H), 2.50 (d, J=5.6 Hz, J=12.8 Hz, 1H), 2.19-2.13 (m,1H), 1.87-1.76 (m, 2H), 1.74-1.62 (m, 3H), 1.47-1.31 (m, 2H), 1.21 (s,3H), 1.17 (s, 3H), 1.15-1.03 (m, 1H), 0.95 (t, J=7.6 Hz, 1H) ppm; Massspectrum (ESI+ve) m/z 193 (M+H⁺).

Example 77: (±)-(3aR,7aS)-4,4,7,7-tetramethyloctahydro-1H-inden-2-ol

To a mixture of the product of Example 70 (15 mg, 0.08 mol) in methanol(3 mL) at room temperature was added sodium borohydride (6 mg, 0.16mmol) and the reaction was stirred for 2 hours. The reaction was dilutedwith ethyl acetate (60 mL) and the organic phase was washed with water(20 mL) and brine (20 mL), dried over anhydrous sodium sulfate andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography to give the title compound as white solid (12mg, Yield: 76%). Mp=106.2-108° C.; R_(f)=0.6 (10:1 petroleum ether/ethylacetate); ¹H NMR (400 MHz, CDCl3) δ 4.38-4.34 (m, 1H), 2.08-2.00 (m,2H), 1.68-1.62 (m, 2H), 1.51-1.37 (m, 5H), 1.29-1.22 (m, 2H), 0.83 (s,6H), 0.80 (s, 6H); ¹³C NMR (100 MHz, CDCl3) δ 72.99, 47.11, 37.17,34.08, 31.60, 31.01, 27.45 ppm; Mass spectrum (El+ve) m/z 196 (M⁺).

Example 78: 4,4,7,7-tetramethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-oneoxime

To a mixture of the product of Example 41 (30 mg, 0.16 mmol) in pyridine(1 mL) at room temperature was added hydroxylamine hydrochloride (33 mg,0.47 mmol) and the reaction was warmed to 160° C. and stirred for 1 hourin a microwave reactor. The reaction was diluted with ethyl acetate theorganic phase was washed with water (20 mL) and brine (20 mL) dried overanhydrous sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to afford thetitle compound as a white solid (11 mg, Yield: 33%). Mp=173.2-175.8° C.;R_(f)=0.6 (10:1 petroleum ether/ethyl acetate); ¹H NMR (400 MHz, CDCl₃)δ 5.88 (s, 1H), 2.65-2.60 (m, 2H), 2.50-2.45 (m, 1H), 1.61-1.29 (m, 4H),1.18 (s, 3H), 1.10 (s, 3H), 0.99 (s, 3H), 0.62 (s, 3H) ppm; Massspectrum (ESI+ve) m/z 208 (M+H⁺).

Example 79:(±)-(3aR,7aS)-4,4,7,7-tetramethylhexahydro-1H-inden-2(3H)-one oxime

To a mixture of the product of Example 70 (30 mg, 0.16 mmol) in pyridine(1 mL) at room temperature was added hydroxylamine hydrochloride (32 mg,0.46 mmol) and the reaction was warmed to 160° C. and stirred for 20minutes in a microwave reactor. The reaction was diluted with ethylacetate the organic phase was washed with water (20 mL) and brine (20mL), dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatographyto afford the title compound as a white solid (25 mg, Yield: 77%).Mp=117.1-119° C.; R_(f)=0.6 (10:1 petroleum ether/ethyl acetate); ¹H NMR(400 MHz, CDCl₃+D₂O) δ 7.60 (s, 1H), 2.44-2.34 (m, 4H), 1.90 (d, J=6.0Hz, 2H), 1.39-1.28 (m, 4H), 0.96 (s, 3H), 0.95 (s, 3H), 0.85 (s, 3H)0.82 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 210 (M+H⁺).

Biology Examples

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

The invention is described in more detail in the following non-limitingexamples. It is to be understood that these particular methods andexamples in no way limit the invention to the embodiments describedherein and that other embodiments and uses will no doubt suggestthemselves to those skilled in the art.

Reagents

Monoclonal anti-rhodopsin 1 D4 antibody can be purchased from Universityof British Columbia.

Cell Lines and Culture Conditions

Stable cell lines expressing opsin protein were generated using theFlp-In T-Rex system. The stable cells were grown in DMEM high glucosemedia supplemented with 10% (v/v) fetal bovine serum,antibiotic/antimycotic solution, 5 μ/ml blasticidin and hygromycin at37° C. in presence of 5% CO₂. For all the experiments the cells wereallowed to reach confluence and were induced to produce opsin with 1μg/mL tetracycline after change of media and then compounds were added.The plates were incubated for 48 hours after which the cells wereharvested.

SDS-PAGE and Western Blotting

Proteins were separated on SDS-PAGE gels and western blotted asdescribed in (Noorwez et al., J. Biol. Chem. 279, 16278-16284 (2004)).

The in vivo efficacy of the compounds of the invention in treatingmacular degeneration can be demonstrated by various tests well known inthe art. For example, human patients are selected based on a diagnosisof macular degeneration (such as where there is a gross diagnosis ofthis condition or where they have been shown to exhibit build-up oftoxic visual cycle products, such as A2E, lipofuscin, or drusen in theireyes. A compound of the invention, such as that of Formula I, isadministered to a test group while a placebo, such as PBS or DMSO, isadministered to a control group that may be as large or may be somewhatsmaller than the test group. The test compound is administered either ona one time basis or on a sequential basis (for example, weekly or daily)or according to some other predetermined schedule.

Administration of the test compound is normally by oral or parenteralmeans and in an amount effective to retard the development and/orreoccurrence of macular degeneration. An effective dose amount isgenerally in the range of about 1 to 5,000 mg or in the range of 10 to2,000 mg/kg. Administration may include multiple doses per day.

Efficacy of the test compound in retarding progression of maculardegeneration is generally by measuring increase in visual acuity (forexample, using Early Treatment Diabetic RP Study (ETDRS) charts(Lighthouse, Long Island, N.Y.). Other means of following and evaluatingefficacy is by measuring/monitoring the autofluorescence or absorptionspectra of such indicators as N-retinylidene-phosphatidylethanolamine,dihydro-N-retinylidene-N-retinyl-phosphatidylethanolamine,N-retinylidene-N-retinyl-phosphatidylethanolamine,dihydro-N-retinylidene-N-retinyl-ethanolamine, and/orN-retinylidene-phosphatidylethanolamine in the eye of the patient.Autofluorescence is monitored using different types of instrument, forexample, a confocal scanning laser ophthalmoscope.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is acommon pathological feature observed in various degenerative diseases ofthe retina. A toxic vitamin A-based fluorophore (A2E) present withinlipofuscin granules has been implicated in death of RPE andphotoreceptor cells. Such experiments can employ an animal model thatmanifests accelerated lipofuscin accumulation to evaluate the efficacyof a therapeutic approach based upon reduction of serum vitamin A(retinol). Administration of test compound to mice harboring a nullmutation in the Stargardt's disease gene (ABCA4) produces reductions inserum retinol/retinol binding protein and arrested accumulation of A2Eand lipofuscin autofluorescence in the RPE.

Test animals are available for use in testing efficacy of a testcompound in reducing build-up of toxic pigments, such as lipofuscin. Forexample, mice have been produced that exhibit increased production ofsuch toxic product. Such mice have been described in the literature(see, for example, Widder et al., U.S. Pub. 2006/0167088) and theirvalue and utility are well known to those in the art.

Showing the efficacy of compounds of the invention in protecting againstlight toxicity is conveniently performed by methods well known in theart (see, for example, Sieving et al, PNAS, Vol. 98, pp 1835-40 (2001)).

Biology Example 1 Rhodopsin Purification and Regeneration

P23H cells were grown to confluency in 10 centermeter plates in DMEMcontaining high glucose, blasticidin (5 μg/ml) and hygromycin (100μg/ml). The cells were induced with tetracycline (1 μg/ml) and treatedwith either DMSO (vehicle) or different concentrations of the test (0.3μM, 1 μM, 3 μM, 10 μM, 30 μM and 80 μM). After 24 hours, the medium wasremoved and fresh medium with the the compounds was added to the plates.β-Ionone (20 μM) was used as a positive control for the experiments. Thecells were harvested 48 hours after the first treatment. All proceduresfrom hereon were carried out under a dim red light (>660 nm). The cellswere washed twice with PBS, and incubated for 1 hour at room temperaturein 1 mL of PBS containing 9-cis-retinal (20 μM). After regeneration, thecells were washed with PBS and incubated for 1 hour at 4° C. in PBScontaining 1% n-dodecyl-β-D maltoside and protease inhibitors (Roche)for lysis. The cell lysate was centrifuged in a tabletop Beckmanultracentrifuge at 36,000×g for 10 minutes. The supernatant was removedand protein was estimated in all of the samples (DC protein assay,Biorad). Equal amounts of protein (5 μg) was loaded on previouslyprepared 1D4-coupled cyanogen bromide-activated Sepharose 4B beads for 1hour at 4° C. Briefly, the Sepharose 4B beads were conjugated with 1D4antibody that recognizes the C-terminus of opsin. The beads wereextensively washed three times with PBS and twice with sodium phosphatebuffer (10 mM, pH 6.0), both containing 0.1% n-dodecyl-β-D maltoside.The protein was eluted in the sodium phosphate buffer containing asynthetic 9 amino acid peptide corresponding to the C-terminus of opsinprotein. The eluted rhodopsin was analyzed on a spectrophotometerscanning the UV-visible range from 250 to 650 nm at increments of 1 nm.

Table 1 contains the results of β-ionone (reference compound 1) and testcompounds in which the 480-500 nm absorbance is expressed as a foldincrease over the DMSO control. FIG. 1 is the spectral results usingβ-ionone according to Biology Example 1.

TABLE 1 Compound Fold Increase Over Control Concentration (μM) β-ionone2.4 20  1 1.7 10  3 2.2 20  5 1.7 10  8 2.1 20 13 2.2 20 14 2.4 20 162.2 20 18 1.7 20 20 1.9 20 22 1.8 20 26 2.3 20 27 2.0 10 28 1.7 10 292.4 10 30 1.8 10 31 1.6 10 33 1.8 10 34 2.2 10 36 2.0 10 37 1.8 10 401.8 10 41 1.9 10 43 1.7 10 44 2.2 10 45 3.0 10 46 1.8 10 51 1.7 10 531.7 10 55 2.8 10 56 1.7 10 58 2.0 20 60 2.1 20 61 1.8 20 62 2.3 20 632.3 20 64 2.1 20 65 1.8 20 66 1.9 20 67 1.8 20 69a 2.0 20 69b 1.8 20 701.9 20 71 2.2 20 72 1.9 20

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A compound of Formula I,

wherein R¹ and R² are independently: 1) hydrogen, 2) —CH₃, or 3)—CH₂CH₃; R_(a) and R_(h) are each independently: 1) hydrogen, 2) —CH₃,or 3) —CH₂CH₃; R^(3′) and R⁵ are each independently: 1) hydrogen, or 2)lower alkyl; and R⁴ is: 1) hydrogen, 2) cycloalkyl, or 3) phenyl;wherein if either R¹ or R² is hydrogen then each of R_(a) and R_(b) isselected from methyl and ethyl, including pharmaceutically acceptablesalts, solvates and hydrates thereof. 2.-4. (canceled)
 5. The compoundof claim 1, wherein each of R¹ and R² is independently methyl or ethyland each of R_(a) and R_(b) is independently hydrogen or methyl.
 6. Thecompound of claim 5, wherein each of each of R¹ and R² is methyl andeach of R_(a) and R_(b) is hydrogen. 7.-49. (canceled)
 50. A method oftreating or preventing an ophthalmic condition in a subject at riskthereof, comprising administering to the subject an effective amount ofa compound of Formula I

wherein R¹ and R² are independently: 1) hydrogen, 2) —CH₃; or 1)—CH₂CH₃; R_(a) and R_(b) are each independently: 1) hydrogen, 2) CH₃, or3) —CH₂CH₃; R^(3′) and R⁵ are each independently: 1) hydrogen, or 2)lower alkyl; and R⁴ is: 1) hydrogen, 2) cycloalkyl, or 3) phenyl;wherein if either R¹ or R² is hydrogen then each of R_(a) and R_(h) isselected from methyl and ethyl, including pharmaceutically acceptablesalts, solvates and hydrates thereof.
 51. The method of claim 50,wherein said ophthalmic condition is an ocular protein mislocalizationdisorder.
 52. The method of claim 50, wherein said ophthalmic conditionis selected from the group consisting of wet or dry age related maculardegeneration (ARMD), retinitis pigmentosa (RP), a retinal or maculardystrophy, Stargardt's disease, Sorsby's dystrophy, autosomal dominantdrusen, Best's dystrophy, peripherin mutation associate with maculardystrophy, dominant form of Stargart's disease, North Carolina maculardystrophy, light toxicity, normal vision loss related aging and normalloss of night vision related to aging.
 53. The method of claim 50,wherein said ophthalmic condition is retinitis pigmentosa (RP).
 54. Themethod of claim 53, wherein said RP is caused by aberrant opsin-folding.55.-80. (canceled)
 81. A method of treating or preventing an ophthalmiccondition in a subject at risk thereof, comprising administering to thesubject an effective amount of a compound of selected from the groupconsisting of: Name Structure 7,7-dimethyl-5,6,7,7a-tetrahydro-1H-inden-2(4H)-one

(−)-4,4,7,7-tetramethyl-5,6,7,7a- tetrahydro-1H-inden-2(4H)-one

(+)-4,4,7,7-tetramethyl-5,6,7,7a- tetrahydro-1H-inden-2(4H)-one

4,4-dimethyl-5,6,7,7a-tetrahydro- 1H-inden-2(4H)-one

4,4,7,7-tetramethyl-5,6,7,7a- tetrahydro-1H-inden-2(4H)-one

(±)-(3aS,7aS)-3a,7,7- trimethylhexahydro-1H-inden- 2(3H)-one

(±)-(3aR,7aS)-3,3a,7,7-tetramethyl- 3a,4,5,6,7,7a-hexahydro-1H-inden-1-one oxime

2,7,7-trimethyl-2,3,4,5,6,7- hexahydro-1H-isoindol-1-one

(±)-(3aR,7aS)-3,3a,7,7-tetramethyl- 3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

(±)-(3R,3aR)-3,4,4-trimethyl- 2,3,3a,4,5,6-hexahydro-1H-inden- 1-one

(3aR,7aS)-4,4,7,7- tetramethylhexahydro-1H-inden- 2(3H)-one

(±)-(1R,7aS)-1,4,4-trimethyl- 5,6,7,7a-tetrahydro-1H-inden- 2(4H)-one

(±)-(1R,3aR,7aS)-2,3,3a,7,7- pentamethyl-3a,4,5,6,7,7a- hexahydro 1Hinden-1-ol

(±)-(3aS,7aS)-2,3,4,4-tetramethyl- 3a,4,5,6,7,7a-hexahydro-1H-inden-1-one

(±)-(3aS,7aS)-3a,7,7- trimethyloctahydro-1H-inden-2-ol

(±)-(3aS,7aS)-3a,7,7- trimethylhexahydro-1H-inden- 2(3H)-one oxime

or a pharmaceutically acceptable salt thereof.